Compositions capable of specifically binding particular human antigen presenting molecule/pathogen-derived antigen complexes and uses thereof

ABSTRACT

A composition-of-matter comprising an antibody or antibody fragment including an antigen-binding region capable of specifically binding an antigen-presenting portion of a complex composed of a human antigen-presenting molecule and an antigen derived from a pathogen is disclosed.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.11/074,803, filed on Mar. 9, 2005, which is a Continuation of U.S.patent application Ser. No. 10/396,578, filed on Mar. 26, 2003, nowabandoned. The contents of the above Applications are all incorporatedherein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to compositions-of-matter capable ofspecifically binding particular antigen-presenting molecule(APM)/antigen complexes. More particularly, the present inventionrelates to compositions-of-matter capable of specifically binding aparticular human APM/pathogen-derived antigen complex.

Diseases caused by pathogens, such as viruses, mycoplasmas, bacteria,fungi, and protozoans, account for a vast number of diseases, includinghighly debilitating/lethal diseases, affecting all human individuals atnumerous instances during their lifetime. For example, diseases causedby retroviruses are associated with various immunological, neurological,and neoplastic disorders. For example, diseases caused by lymphotropicretroviruses, such as acquired immunodeficiency syndrome (AIDS) causedby human immunodeficiency virus (HIV), or the closely related humanT-cell lymphotropic virus (HTLV), a causative agent of various lethalpathologies (for general references, refer, for example to: Johnson J M.et al., 2001. Int J Exp Pathol. 82:135-47; and Bangham C R., 2000. JClin Pathol. 53:581-6), account for lethal disease epidemics ofdevastating human and economic impact.

However, satisfactory methods of diagnosing, characterizing, andtreating many kinds of pathogen-associated diseases such as diseasesassociated with lymphotropic viruses such as HIV or HTLV areunavailable.

HTLV-1 was the first human retrovirus identified (Poiesz B. J. et al.,1980. Proc Natl Acad Sci USA. 77:7415-7419). It causes a variety ofdiseases, including adult T lymphocyte leukemia/lymphoma (ATLL; YoshidaM. et al., 1982. Proc Natl Acad Sci USA. 79:2031-2035) and a nonneoplasic inflammatory neurological syndrome called human T lymphotropictype I (HTLV-I)-associated myelopathy/tropical virus spastic paraparesis(HAM/TSP; Osame M. et al., 1986. Lancet 1:1031-1032). Several otherclinical conditions have been linked to HTLV-1 infection on the basis ofseroepidemiological studies; these include Sjogren's syndrome,inflammatory arthropathies, polymyositis, and pneumopathies (Coscoy L.et al., 1998. Virology 248: 332-341). The HTLV protein Tax seems to playa major role in the pathogenesis of HTLV-I associated diseases. Taxprotein is known to stimulate the transcription of viral and cellulargenes such as the genes coding for interleukin-2 (IL-2) and othercytokines, interleukin-2 receptor (IL-2R), proto-oncogenes, c-jun andc-fos, and MHC molecules (Yoshida M., 1993. Trends Microbiol.1:131-135). The transforming potential of Tax has been demonstrated indifferent experimental systems. It has been shown that rodentfibroblastic cell lines expressing Tax form colonies in soft agar andtumors in nude mice (Tanaka A. et al., 1990. Proc Natl Acad Sci USA.87:1071-1075). Also, Tax transforms primary fibroblasts in cooperationwith the Ras protein (Pozzatti R. et al., 1990. Mol Cell Biol.10:413-417), and immortalizes primary T lymphocytes in the presence ofIL-2 (Grassmann R. et al., 1989. Proc Natl Acad Sci USA. 86:3351-3355).Transgenic mice carrying the tax gene develop different types of tumors(Grossman W. J. et al., 1995. Proc Natl Acad Sci USA. 92:1057-1061). Taxbinds directly to DNA but acts in cooperation with several cellulartranscription factors, but the role of these different interactions inthe cell transformation mediated by Tax is still unclear (Coscoy L. etal., 1998. Virology 248: 332-341). HTLV-1 associated myelopathy is aslowly progressive neurological disease characterized by inflammatoryinfiltrates in the central nervous system that consist predominantly ofmonocytes and CD8 positive T lymphocytes. Systemically, there is anincrease in viral load associated with clonal expansion of HTLV-1reactive CD8 positive T lymphocytes which are primarily reactive withthe Tax protein. It has been shown that in patients carrying the HLA-A2allele, the immune response is dominated by CD8 positive T lymphocytesthat recognize the Tax₁₁₋₁₉ peptide (Bieganowska K. et al., 1999. J.Immunol. 162:1765-1771; Nagai, M. et al., 2001. J Inf Dis. 183:197-205).

The immune system employs two types of immune responses to provideantigen specific protection from pathogens; humoral immune responses,and cellular immune responses, which involve specific recognition ofpathogen antigens via antibodies and T lymphocytes, respectively.

T lymphocytes, by virtue of being the antigen specific effectors ofcellular immunity, play a central and direct role in the body's defenseagainst diseases mediated by intracellular pathogens, such as viruses,intracellular bacteria, mycoplasmas, and intracellular parasites, bydirectly cytolysing cells infected by such pathogens. However, helper Tlymphocytes also play a critical role in humoral immune responsesagainst non intracellular pathogens by providing T cell help to Blymphocytes in the form of interleukin secretion to stimulate productionof antibodies specific for antigens of such pathogens.

The specificity of T lymphocyte responses is conferred by, and activatedthrough T-cell receptors (TCRs). T-cell receptors are antigen specificreceptors clonally distributed on individual T lymphocytes whoserepertoire of antigenic specificity is generated via somatic generearrangement mechanisms analogously to those involved in generating theantibody gene repertoire. T-cell receptors are composed of a heterodimerof transmembrane molecules, the main type being composed of an αβ dimerand a smaller subset of a γδ dimer. T lymphocyte receptor subunitscomprise a transmembrane constant region and a variable region in theextracellular domain, similarly to immunoglobulins, and signaltransduction triggered by TCRs is indirectly mediated via CD3/ζ, anassociated multi-subunit complex comprising signal transducing subunits.

The two main classes of T lymphocytes, helper T lymphocytes andcytotoxic T lymphocytes (CTLs), are distinguished by expression of thesurface markers CD4 and CD8, respectively. As described hereinabove, themain function of helper T lymphocytes is to secrete cytokines, such asIL-2, promoting activation and proliferation of CTLs and B lymphocytes,and the function of CTLs is to induce apoptotic death of cellsdisplaying immunogenic antigens.

T lymphocyte receptors, unlike antibodies, do not recognize nativeantigens but rather recognize cell-surface displayed complexescomprising an intracellularly processed fragment of a protein or lipidantigen in association with a specialized antigen-presenting molecule(APM): major histocompatibility complex (MHC) for presentation ofpeptide antigens; and CD1 for presentation of lipid antigens, and to alesser extent, peptide antigens. Peptide antigens displayed by MHCmolecules and lipid antigens displayed by CD1 molecules havecharacteristic chemical structures are referred to as MHC-restrictedpeptides and CD1 restricted lipids, respectively. Majorhistocompatibility complex molecules are highly polymorphic, comprisingmore than 40 common alleles for each individual gene. “Classical” MHCmolecules are divided into two main types, class I and class II, havingdistinct functions in immunity.

Major histocompatibility complex class I molecules are expressed on thesurface of virtually all cells in the body and are dimeric moleculescomposed of a transmembrane heavy chain, comprising the peptide antigenbinding cleft, and a smaller extracellular chain termedβ₂-microglobulin. MHC class I molecules present 9- to 11-amino acidresidue peptides derived from the degradation of cytosolic proteins bythe proteasome a multi-unit structure in the cytoplasm, (Niedermann G.,2002. Curr Top Microbiol Immunol. 268:91-136; for processing ofbacterial antigens, refer to Wick M J, and Ljunggren H G., 1999. ImmunolRev. 172:153-62). Cleaved peptides are transported into the lumen of theendoplasmic reticulum (ER) by TAP where they are bound to the groove ofthe assembled class I molecule, and the resultant MHC/antigen complex istransported to the cell membrane to enable antigen presentation to Tlymphocytes (Yewdell J W., 2001. Trends Cell Biol. 11:294-7; Yewdell JW. and Bennink J R., 2001. Curr Opin Immunol. 13:13-8).

Major histocompatibility complex class II molecules are expressed on arestricted subset of specialized antigen-presenting cells (APCs)involved in T lymphocyte maturation and priming. Such APCs in particularinclude dendritic cells and macrophages, cell types which internalize,process and display antigens sampled from the extracellular environment.Unlike MHC class I molecules, MHC class II molecules are composed of anαβ transmembrane dimer whose antigen binding cleft can accommodatepeptides of about 10 to 30, or more, amino acid residues.

The antigen presenting molecule CD1, whose main function, as describedhereinabove, is presentation of lipid antigens, is a heterodimercomprising a transmembrane heavy chain paired with β₂-microglobulin,similarly to MHC class I, and is mainly expressed on professional APCs,similarly to MHC class II (Sugita M. and Brenner M B., 2000. SeminImmunol. 12:511). CD1/antigen complexes are specifically recognized byTCRs expressed on CD4⁻CD8⁻ T lymphocytes and NKT lymphocytes and play asignificant role in microbial immunity, tumor immunology, andautoimmunity.

The cells of the body are thus scanned by T lymphocytes during immunesurveillance or during maturation of T lymphocytes for theirintracellular protein or lipid content in the form of such APM/antigencomplexes.

One strategy which has been proposed to enable optimal diagnosis,characterization, and treatment of diseases associated with an infectionby a pathogen involves using molecules capable of specifically bindingAPM/antigen complexes composed of a particular combination of APM and anantigen derived from such a pathogen. Such molecules, for example, couldbe conjugated to functional moieties, such as detectable moieties ortoxins, and the resultant conjugates could be used to detect suchcomplexes or cells displaying such complexes, or to kill cellsdisplaying such complexes. Hence, such conjugates could be used todiagnose/characterize and treat a pathogen infection in an individual,respectively. Alternately, molecules capable of specifically bindingsuch complexes could be used to bind such complexes on cells so as toblock activation of T lymphocytes bearing TCRs specific for suchcomplexes. Such molecules could further be used, for example, to isolatesuch complexes, or cells displaying such complexes, such as cellsinfected with a pathogen, or APCs exposed to a pathogen-derived antigen.

Several prior art approaches have been described involving moleculescapable of binding particular APM/antigen complexes.

One approach involves using TCRs or derivatives thereof specific forparticular MHC/peptide complexes in attempts to provide reagents capableof specifically binding such complexes.

Another approach involves using antibodies or derivatives thereofspecific for particular mouse MHC/peptide complexes in attempts toprovide reagents capable of specifically binding such complexes(Aharoni, R. et al., 1991. Nature 351:147-150; Andersen, P. S. et al.,1996. Proc. Natl. Acad. Sci. U.S.A 93:1820-1824; Dadaglio, G. et al.,1997. Immunity 6:727-738; Day, P. M. et al., 1997. Proc. Natl. Acad.Sci. U.S. A. 94:8064-8069; Krogsgaard, M. et al., 2000. J. Exp. Med.191:1395-1412; Murphy, D. B. et al., 1989. Nature 338:765-768; Porgador,A. et al., 1997. Immunity 6:715-726; Reiter, Y. et al., Proc. Natl.Acad. Sci. U.S.A. 94:4631-4636; Zhong, G. et al., 1997. Proc. Natl.Acad. Sci. U.S.A. 94:13856-13861; Zhong, G. et al., 1997. J. Exp. Med.186:673-682).

A further approach involves utilizing antibodies or derivatives thereofspecific for the human MHC class I molecule HLA-A1 in complex with anHLA-A1 restricted peptide derived from the melanoma specific tumorassociated antigen melanoma associated antigen (MAGE)-A1 in attempts toprovide reagents capable of specifically binding such a complex (Chames,P. et al., 2000. Proc. Natl. Acad. Sci. U.S.A. 97:7969-7974).

An additional approach involves employing antibodies or derivativesthereof specific for the human MHC class I molecule HLA-A2 in complexwith an HLA-A2 restricted peptide derived from the melanoma specifictumor associated antigen gp100 in attempts to provide reagents capableof specifically binding such a complex (Denkberg, G. et al., 2002. Proc.Natl. Acad. Sci. U.S.A. 99:9421-9426).

Yet another approach involves using antibodies or derivatives thereofspecific for human MHC class I molecule HLA-A2 in complex with an HLA-A2restricted peptide derived from human telomerase catalytic subunit(hTERT) in attempts to provide reagents capable of specifically bindingsuch a complex (Lev, A. et al., 2002. Cancer Res. 62:3184-3194).

However, all of the aforementioned prior art approaches suffer fromsignificant disadvantages: (i) approaches involving the use TCRs orportions thereof as compounds capable of specifically binding particularMHC/peptide complexes are suboptimal due to the relatively low intrinsicbinding affinity of TCRs for such complexes; (ii) approaches involvingthe use of antibodies or portions thereof specific for MHC/peptidecomplexes comprising non-human MHC are not suitable for humanapplication; and (iii) approaches involving antibodies or portionsthereof specific for MHC/non-pathogen derived antigen complexes are notsuitable for specifically binding complexes comprising pathogen derivedantigens.

Thus, all prior art approaches have failed to provide an adequatesolution for providing molecules capable of specifically binding withhigh specificity and affinity a particular human APM/pathogen-derivedantigen complex.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, molecules devoid of the above limitation.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of detecting an antigen-presenting portion of a complex composedof a human antigen-presenting molecule and an antigen derived from apathogen, the method comprising: (a) exposing the antigen-presentingportion of the complex to a composition-of-matter comprising an antibodyor antibody fragment including an antigen-binding region capable ofspecifically binding the antigen-presenting portion of the complex, tothereby obtain a conjugate of the antigen-presenting portion of thecomplex and the antibody or antibody fragment; and (b) detecting theantibody or antibody fragment of the conjugate, thereby detecting anantigen-presenting portion of a complex composed of a humanantigen-presenting molecule and an antigen derived from a pathogen.

According to further features in preferred embodiments of the inventiondescribed below, the complex is displayed or expressed by a target cell,and step (a) is effected by exposing the target to thecomposition-of-matter.

According to still further features in the described preferredembodiments the method of detecting an antigen-presenting portion of acomplex composed of a human antigen-presenting molecule and an antigenderived from a pathogen further comprises: (c) obtaining the target cellfrom an individual.

According to another aspect of the present invention there is provided amethod of detecting in a biological sample an antigen-presenting portionof a complex composed of an antigen-presenting molecule and an antigen,the method comprising: (a) attaching the biological sample to a surface;(b) exposing the biological sample to a composition-of-matter comprisingan antibody or antibody fragment including an antigen-binding regioncapable of specifically binding the antigen-presenting portion of thecomplex, to thereby obtain a conjugate of the antigen-presenting portionof the complex and the antibody or antibody fragment; and (c) detectingthe antibody or antibody fragment of the conjugate, thereby detecting ina biological sample an antigen-presenting portion of a complex composedof a human antigen-presenting molecule and an antigen.

According to further features in preferred embodiments of the inventiondescribed below, the method of detecting in a biological sample anantigen-presenting portion of a complex composed of anantigen-presenting molecule and an antigen further comprises: (d)obtaining the biological sample from an individual.

According to still further features in the described preferredembodiments, step (b) is effected by administering thecomposition-of-matter to an individual.

According to still further features in the described preferredembodiments, the antigen is derived from a pathogen.

According to still further features in the described preferredembodiments, the biological sample is infected with the pathogen.

According to still further features in the described preferredembodiments, the biological sample is a cell sample or a tissue sample.

According to yet another aspect of the present invention there isprovided a method of diagnosing an infection by a pathogen in anindividual, the method comprising: (a) exposing a target cell of theindividual to a composition-of-matter comprising an antibody or antibodyfragment including an antigen-binding region capable of specificallybinding an antigen-presenting portion of a complex composed of a humanantigen-presenting molecule and an antigen derived from the pathogen, tothereby obtain a conjugate of the antigen-presenting portion of thecomplex and the antibody or antibody fragment; and (b) detecting theantibody or antibody fragment of the conjugate, thereby diagnosing aninfection by a pathogen in an individual.

According to further features in preferred embodiments of the inventiondescribed below, the method of diagnosing an infection by a pathogen inan individual further comprises: (c) obtaining the target cell from theindividual.

According to still further features in the described preferredembodiments, step (a) is effected by administering thecomposition-of-matter to the individual.

According to still further features in the described preferredembodiments, the composition-of-matter further comprises a detectablemoiety attached to the antibody or antibody fragment, and detecting theantibody or antibody fragment of the conjugate is effected by detectingthe detectable moiety attached to the antibody or antibody fragment ofthe conjugate.

According to still another aspect of the present invention there isprovided a method of killing or damaging a target cell expressing ordisplaying an antigen-presenting portion of a complex composed of ahuman antigen-presenting molecule and an antigen derived from apathogen, the method comprising exposing the target cell to acomposition-of-matter comprising an antibody or antibody fragmentincluding an antigen-binding region capable of specifically binding theantigen-presenting portion of the complex, thereby killing or damaging atarget cell expressing or displaying an antigen-presenting portion of acomplex composed of a human antigen-presenting molecule and an antigenderived from a pathogen.

According to further features in preferred embodiments of the inventiondescribed below, the method of killing or damaging a target cellexpressing or displaying an antigen-presenting portion of a complexcomposed of a human antigen-presenting molecule and an antigen derivedfrom a pathogen further comprises the step of obtaining the target cellfrom an individual.

According to still further features in the described preferredembodiments, exposing the target cell to the composition-of-matter iseffected by administering the composition-of-matter to an individual.

According to still further features in the described preferredembodiments, the target cell is infected with the pathogen.

According to still further features in the described preferredembodiments, the target cell is a T lymphocyte or an antigen presentingcell.

According to still further features in the described preferredembodiments, the antigen presenting cell is a B cell or a dendriticcell.

According to a further aspect of the present invention there is provideda method of treating a disease associated with a pathogen in anindividual, the method comprising administering to the individual atherapeutically effective amount of a pharmaceutical compositioncomprising as an active ingredient, a composition-of-matter comprisingan antibody or antibody fragment including an antigen-binding regioncapable of specifically binding an antigen-presenting portion of acomplex composed of a human antigen-presenting molecule and an antigenderived from the pathogen, thereby treating a disease associated with apathogen in an individual.

According to a yet a further aspect of the present invention there isprovided an isolated polynucleotide comprising a first nucleic acidsequence encoding an antibody fragment, the antibody fragment includingan antigen-binding region capable of specifically binding anantigen-presenting portion of a complex composed of a humanantigen-presenting molecule and an antigen derived from a pathogen.

According to further features in preferred embodiments of the inventiondescribed below, the isolated polynucleotide further comprises a secondnucleic acid sequence encoding a polypeptide selected from the groupconsisting of a coat protein of a virus, a detectable moiety, and atoxin.

According to still further features in the described preferredembodiments, the second nucleic acid sequence is translationally fusedwith the first nucleic acid sequence.

According to still a further aspect of the present invention there isprovided a nucleic acid construct comprising the isolated polynucleotideand a promoter sequence for directing transcription of the isolatedpolynucleotide in a host cell.

According to further features in preferred embodiments of the inventiondescribed below, the promoter sequence is a T7 promoter sequence.

According to still further features in the described preferredembodiments, the promoter sequence is capable of driving expression ofthe nucleic acid sequence in a prokaryote.

According to still further features in the described preferredembodiments, the promoter sequence is capable of driving inducibleexpression of the nucleic acid sequence.

According to an additional aspect of the present invention there isprovided a host cell comprising the nucleic acid construct.

According to further features in preferred embodiments of the inventiondescribed below, the host cell is a prokaryotic cell.

According to still further features in the described preferredembodiments, the prokaryotic cell is an E. coli cell.

According to yet an additional aspect of the present invention there isprovided a virus comprising the nucleic acid construct.

According to still an additional aspect of the present invention thereis provided a virus comprising a coat protein fused to an antibodyfragment including an antigen-binding region capable of specificallybinding an antigen-presenting portion of a complex composed of a humanantigen-presenting molecule and an antigen derived from a pathogen.

According to further features in preferred embodiments of the inventiondescribed below, the virus is a filamentous phage and the coat proteinis pIII.

According to another aspect of the present invention there is provided acomposition-of-matter comprising an antibody or antibody fragmentincluding an antigen-binding region capable of specifically binding anantigen-presenting portion of a complex composed of a humanantigen-presenting molecule and an antigen derived from a pathogen.

According to yet another aspect of the present invention there isprovided a pharmaceutical compositions comprising as an activeingredient the composition-of-matter and a pharmaceutically acceptablecarrier.

According to still another aspect of the present invention there isprovided a composition-of-matter comprising a multimeric form of anantibody or antibody fragment including an antigen-binding regioncapable of specifically binding an antigen-presenting portion of acomplex composed of a human antigen-presenting molecule and an antigenderived from a pathogen.

According to a further aspect of the present invention there is provideda pharmaceutical composition comprising as an active ingredient thecomposition-of-matter comprising a multimeric form of an antibody orantibody fragment including an antigen-binding region capable ofspecifically binding an antigen-presenting portion of a complex composedof a human antigen-presenting molecule and an antigen derived from apathogen, and a pharmaceutically acceptable carrier.

According to further features in preferred embodiments of the inventiondescribed below, the antibody is a monoclonal antibody.

According to still further features in the described preferredembodiments, the antibody fragment is a monoclonal antibody fragment.

According to still further features in the described preferredembodiments, the antibody fragment is selected from the group consistingof an Fd fragment, an Fab, and a single chain Fv.

According to still further features in the described preferredembodiments, the antigen-binding region includes an amino acid sequenceselected from the group consisting of SEQ ID NOs: 14 to 97.

According to still further features in the described preferredembodiments, the antibody or antibody fragment, or a part of theantibody or antibody fragment is of human origin.

According to still further features in the described preferredembodiments, the part of the antibody or antibody fragment is a portionof a constant region of the antibody or antibody fragment, or a constantregion of the antibody or antibody fragment.

According to still further features in the described preferredembodiments, the binding of the antibody or antibody fragment to theantigen-presenting portion of the complex is characterized by anaffinity having a dissociation constant selected from the rangeconsisting of 1×10⁻² molar to 5×10⁻¹⁶ molar.

According to still further features in the described preferredembodiments, the composition-of-matter further comprises a toxin ordetectable moiety attached to the antibody or antibody fragment.

According to still further features in the described preferredembodiments, the detectable moiety is selected from the group consistingof a recognition sequence of a biotin protein ligase, a biotin molecule,a streptavidin molecule, a fluorophore, an enzyme, and a polyhistidinetag.

According to still further features in the described preferredembodiments, the biotin protein ligase is BirA.

According to still further features in the described preferredembodiments, the fluorophore is phycoerythrin.

According to still further features in the described preferredembodiments, the enzyme is horseradish peroxidase.

According to still further features in the described preferredembodiments, the toxin is Pseudomonas exotoxin A or a portion thereof.

According to still further features in the described preferredembodiments, the portion of Pseudomonas exotoxin A is a translocationdomain and/or an ADP ribosylation domain.

According to still further features in the described preferredembodiments, the human antigen-presenting molecule is a majorhistocompatibility complex molecule.

According to still further features in the described preferredembodiments, the major histocompatibility complex molecule is a majorhistocompatibility complex class I molecule.

According to still further features in the described preferredembodiments, the major histocompatibility complex class I molecule is anHLA-A2 molecule.

According to still further features in the described preferredembodiments, the human antigen-presenting molecule is a single chainantigen-presenting molecule.

According to still further features in the described preferredembodiments, the pathogen is a viral pathogen.

According to still further features in the described preferredembodiments, the viral pathogen is a retrovirus.

According to still further features in the described preferredembodiments, the retrovirus is human T lymphotropic virus-1.

According to still further features in the described preferredembodiments, the antigen derived from a pathogen is restricted by theantigen-presenting molecule.

According to still further features in the described preferredembodiments, the antigen derived from a pathogen is a polypeptide.

According to still further features in the described preferredembodiments, the polypeptide is a segment of a Tax protein, or apolypeptide having an amino acid sequence as set forth in SEQ ID NO: 3.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing a composition-of-mattercomprising an antibody or antibody fragment capable of binding withoptimal specificity/affinity a human APM/pathogen-derived antigencomplex.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a histogram depicting specific binding of recombinantFab-phage clones to HLA-A2/Tax₁₁₋₁₉ complex, as determined by ELISA.TAX—HLA-A2/Tax₁₁₋₁₉ complex, gp100-154—HLA-A2/G9-154 peptide complex,MUC1-D6—HLA-A2/MUC1-D6 peptide complex, MART 27—HLA-A2/MART 27 peptidecomplex.

FIGS. 2 a-c are photographs depicting Western immunoblotting assays ofexpression and purification of Fab's selected for specific binding toHLA-A2/Tax₁₁₋₁₉ complex. Shown are SDS-PAGE analyses of purified Fabprotein after metal affinity chromatography, inclusion bodies from BL21cultures expressing Fab T3F2 light chain and Fd fragment, and purifiedin vitro refolded non-reduced (NR) and reduced (R) Fab T3F2 (FIGS. 2a-c, respectively). M—molecular weight markers.

FIGS. 3 a-c are histograms depicting specific binding of solublepurified Fab's T3D4, T3E3, and T3F2, respectively, to immobilizedHLA-A2/Tax₁₁₋₁₉ complex, but not to HLA-A2/control peptide complexes, asdetermined by ELISA.

FIGS. 4 a-b are data plots depicting the binding characteristics ofFab's T3E3 and T3F2, respectively, as determined by titration ELISAusing single chain HLA-A2/Tax₁₁₋₁₉ complex as binding target.

FIG. 4 c is a competitive binding analysis data plot depicting theability of purified Fab T3F2 to inhibit the binding of [125]iodinelabeled Fab T3F2 to immobilized HLA-A2/Tax complex. The apparent bindingaffinity of the recombinant Fab was determined as the concentration ofcompetitor (soluble purified Fab) required for 50 percent inhibition ofthe binding of the [125]iodine labeled tracer.

FIGS. 5 a-f are flow cytometry histograms depicting specific detectionof HLA-A2/Tax₁₁₋₁₉ complex on the surface of APCs. RMAS-HHD, JY, andhuman dendritic (DC) cells (FIGS. 2 a-b, 2 c-d, and 2 e-f, respectively)were loaded with Tax₁₁₋₁₉ peptide or negative control melanoma gp100derived peptide G9-154, as described in the experimental procedures.Peptide-loaded cells were then incubated with the soluble purifiedHLA-A2/Tax₁₁₋₁₉ complex specific Fab's T3E3 (FIGS. 5 a, 5 c, and 5 e) orT3F2 (FIGS. 5 b, 5 d, and 5 f). Note specific staining of cells loadedwith Tax₁₁₋₁₉ but not negative control peptide. Control unloaded cellsare shown in black trace. Control assays were performed using the 10different negative control HLA-A2 restricted peptides listed underMaterials and Methods.

FIGS. 6 a-c are flow cytometry histograms depicting specific detectionof HLA-A2/Tax₁₁₋₁₉ complex on the surface of antigen-presenting cells(APCs) using Fab T3F2 tetramer. RMAS-HHD, JY, or HLA-A2 positive maturedendritic cells (FIGS. 6 a-c, respectively) were pulsed with Tax₁₁₋₁₉peptide. Peptide pulsed cells were then incubated with phycoerythrinconjugated T3F2 tetramer or monomer, as indicated. Fab monomer bindingwas detected using phycoerythrin conjugated anti human Fab antibody.Control unloaded cells stained with the T3F2 tetramer are shown.

FIGS. 7 a-d depict specific detection of cell surface displayedHLA-A2/Tax₁₁₋₁₉ complex by T3F2 after naturally occurring activeintracellular processing. FIGS. 7 a-b are flow cytometry histogramsdepicting specific detection of HLA-A2/Tax₁₁₋₁₉ complex on the surfaceof HLA-A2 positive JY cells, but not HLA-A2 negative APD cells,respectively. Cells were transfected with pcDNA control vector or withpcDNA containing the intact full length Tax gene (pcTAX), and 12 to 24hours following transfection, cells were stained by flow cytometry usingFab T3F2 or the negative control Fab G2D12 specific for HLA-A2/G9-154complex. FIG. 7 c is a bar graph depicting the efficiency of Tax genetransduction into JY and APD cells, as monitored by transfection of thepcDNA vector carrying the GFP gene. FIG. 7 d is a flow cytometryhistogram depicting staining of HLA-A2 positive RSCD4 and HLA-A2negative HUT102 cells (which are lines of human CD4 positive T-cellsinfected with HTLV-1) with phycoerythrin conjugated Fab T3F2 tetramer,or negative control G2D12, as indicated.

FIGS. 8 a-b depict quantitation of the number of HLA-A2/Tax₁₁₋₁₉complexes on the surface of Tax₁₁₋₁₉ peptide pulsed cells. JY APCs werepulsed with various concentrations of Tax₁₁₋₁₉ peptide and surfacedisplay of HLA-A2-Tax₁₁₋₁₉ peptide complex on the cells was analyzed byflow cytometry using phycoerythrin conjugated T3F2 Fab. FIG. 8 a is abar graph depicting the calculated number of complexes per cell withvarious concentration of peptide. The level of fluorescence intensity onstained cells was quantitated flow cytometrically using calibrationbeads conjugated to graded numbers of phycoerythrin molecules(QuantiBRITE PE beads, Becton-Dickinson). FIG. 8 b is a flow cytometryhistogram depicting fluorescence intensity as a function of Tax₁₁₋₁₉peptide concentration.

FIGS. 8 c-d depict high-sensitivity quantitative detection ofHLA-A2/Tax₁₁₋₁₉ complex on the surface JY APCs transfected with the Taxgene mixed at different ratios within a non-transfected cell population.The mixed population was stained with Fab T3F2 and detection sensitivitywas monitored by single-color flow cytometry. FIG. 8 c is a set ofoverlapping flow cytometry histograms shown in large-scale (left panel)or zoomed (right panel) depicting quantitative detection of transfectedcells mixed into populations of non-transfected cells at the variousratios, as indicated. FIG. 8 d is a data table depicting sensitivity ofdetection of HLA-A2/Tax₁₁₋₁₉ complex as a function of the percentage oftransfected cells admixed within a population of non-transfected cells,on the basis of a transfection efficiency of 62.1 percent. Notedetection of HLA-A2/Tax₁₁₋₁₉ complex-displaying cells present in apopulation of non-transfected cells in a proportion as low as 1 percent.

FIGS. 9 a-f are photomicrographs depicting immunohistochemical detectionof HLA-A2/Tax₁₁₋₁₉ complex by Fab T3F2 following intracellularprocessing. FIGS. 9 a-b depict ×60 and ×40 original magnification views,respectively, of Tax transfected JY cells stained with Fab T3F2. FIG. 9c depicts control non transfected JY cells stained with Fab T3F2. FIG. 9d depicts staining of Tax transfected JY cells with negative control FabG2D12 specific for HLA-A2/G9-154 complex. FIGS. 9 e-f depict HLA-A2negative cells transfected for expression of Tax or not transfected,respectively, stained with T3F2. Cells were adsorbed onto poly-L-lysinecoated glass slips 12 to 24 hours following transfection, and stainedwith Fab T3F2. As a negative control Fab G2D12 was used.

FIG. 10 is a data plot depicting specific and efficient killing oftarget cells displaying a specific human MHC/viral peptide complex by afusion protein consisting of an anti specific human MHC/viral peptidecomplex Fab conjugated to a toxin. A cytotoxicity assay was performedusing T3F2-PE38 KDEL fusion protein, consisting of anti HLA-A2/Tax₁₁₋₁₉complex Fab fused to the PE38 KDEL truncated form of pseudomonasexotoxin A. To assay cytolysis by the fusion protein, JY cells loadedwith Tax₁₁₋₁₉ peptide, loaded with control HLA-A2 restricted peptides,or not peptide loaded were incubated with T3F2-PE38 KDEL. Note specificand efficient T3F2-PE38 KDEL mediated killing of cells loaded withTax₁₁₋₁₉ peptide, but not of control JY cells loaded control peptide, orof JY cells not peptide loaded.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of compositions-of-matter capable ofspecifically binding particular antigen-presenting molecule(APM)/antigen complexes, and to methods of using suchcompositions-of-matter to detect, characterize or kill/damagecells/tissues expressing/displaying such complexes. In particular, thepresent invention can be used to optimally detect, characterize orkill/damage human cells/tissues displaying/expressing a particular humanAPM/pathogen-derived antigen complex, such as cells/tissues infectedwith a pathogen, or antigen-presenting cells (APCs) exposed to thepathogen, or an antigen thereof. As such the compositions-of-matter ofthe present invention can be used, for example, to optimally diagnose,characterize, and treat a pathogen infection in a human.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Molecules capable of binding with optimal specificity/affinity aparticular human APM/pathogen-derived antigen complex would be ofsignificant and unique utility since they would enable optimaldiagnosis, characterization, and treatment of pathogen infections inhumans.

Various molecules capable of binding specific APM/antigen complexes havebeen described by the prior art.

For example, one approach involves using antibodies or derivativesthereof specific for mouse MHC/peptide complexes in attempts to providecompounds capable of specifically binding such murine complexes.

Another approach involves using antibodies or derivatives thereofspecific for human MHC/tumor associated antigen peptide complexes inattempts to provide compounds capable of specifically binding such humantumor antigen-presenting complexes.

A further approach involves using antibodies or derivatives thereofspecific for human MHC/telomerase-derived peptide complexes in attemptsto provide compounds capable of specifically binding such humantelomerase antigen-presenting complexes.

However, all such prior art approaches suffer from significantdrawbacks. Prior art approaches involving molecules capable ofspecifically binding complexes comprising non-human APMs do not haveutility for human applications, and prior art approaches involvingcompositions-of-matter capable of specifically binding complexescomprising non-pathogen derived antigens do not have utility forapplications requiring molecules capable of specifically bindingcomplexes comprising pathogen-derived antigens, such as diagnosis,characterization, and treatment of pathogen infections in humans.

Thus, the prior art has failed to provide molecules capable of bindingparticular human APM/pathogen-derived antigen complexes with optimalspecificity and affinity.

While reducing the present invention to practice molecules capable ofbinding particular human APM/pathogen-derived antigen complexes withoptimal specificity and affinity were unexpectedly uncovered. Such acapacity is unique relative to all prior art molecules capable ofbinding particular APM/antigen complexes.

It was also unexpectedly uncovered that attaching such molecules to adetectable moiety or toxin could be used, respectively, todetect/characterize, or kill/damage with optimal efficiency/specificitycells/tissues displaying such complexes. Such capacities are also uniquerelative to all prior art molecules capable of binding particularAPM/antigen complexes.

Thus, in sharp contrast to prior art molecules capable of bindingparticular APM/antigen complexes, the molecules of the present inventioncan be used to detect, or characterize with optimal specificity andsensitivity, or kill with optimal efficiency and specificity humancells/tissues infected with a pathogen, or antigen-presenting cellsexposed to a pathogen, or an antigen thereof.

Thus, according to one aspect of the present invention there is provideda composition-of-matter comprising an antibody or antibody fragmentincluding an antigen-binding region capable of specifically binding anantigen-presenting portion of a complex composed of an APM and anantigen derived from a pathogen.

The composition-of-matter is optimal for use in essentially anyapplication benefiting from a reagent having the capacity tospecifically bind the antigen-presenting portion of a complex composedof a particular APM and a particular antigen derived from a pathogenwhich is restricted by such an APM (referred to hereinafter as “complex”or “the complex”). Such applications particularly include thoseinvolving: (i) specific detection of the antigen-presenting portion ofthe complex; (ii) killing/damaging cells/tissues displaying/expressingthe antigen-presenting portion of the complex (referred to herein as“target cells/tissues”), including pathogen-infected cells or APCsexposed to an antigen of the pathogen; and (iii) blocking binding of theantigen-presenting portion of the complex to a cognate T-cell receptor(TCR); and (iv) and isolating the complex or a celldisplaying/expressing the complex.

As used herein, the term “antibody” refers to a substantially whole orintact antibody molecule.

As used herein, the phrase “antibody fragment” refers to moleculecomprising a portion or portions of an antibody capable of specificallybinding an antigenic determinant or epitope, such as theantigen-presenting portion of the complex.

As used herein, the phrase “antigen-binding region”, when relating tothe antibody or antibody fragment, refers to a portion of the antibodyor antibody or antibody fragment (typically a variable portion) capableof specifically binding a particular antigenic determinant or epitope,or particular set of antigenic determinants or epitopes.

As used herein, the term “APM” refers to an antigen-presenting moleculesuch as an MHC molecule, a CD1 molecule, and a molecule structurallyand/or functionally analogous to an MHC or CD1 molecule. A specific APMis typically capable of binding any of a particular set of distinctantigens so as to form an antigen-presenting complex therewith which canbe specifically bound by a variable portion of a TCR. Antigen-presentingmolecules forming complexes whose antigen-presenting portions compriseantigenic determinants or epitopes which can be specifically bound bythe antibody or antibody fragment comprised in the composition-of-matterare described in further detail hereinbelow.

As used herein, the term “antigen” refers to a molecule or portionthereof (typically a peptide or a lipid), where such a molecule orportion thereof is capable of specifically binding an antigen-bindinggroove of an APM. Such an antigen is commonly referred to in the art asbeing “restricted” by such an APM. A typical antigen, such as apathogen-derived antigen, is typically generated in a human cell byintracellular processing of a larger molecule derived from the pathogen.Such cells typically include a cell infected with the pathogen—inparticular an intracellular pathogen, or an APC exposed to an antigenderived from the pathogen. The antigen generally has a characteristicdimension and/or chemical composition—for example, a characteristicamino acid length and set of anchor residues, respectively, in the caseof a peptide antigen-enabling it to specifically bind theantigen-binding groove of a particular APM so as to form an APM/antigencomplex therewith having an antigen presenting portion capable ofspecifically binding a variable region of a cognate TCR.

As used herein, the phrase “antigen-presenting portion”, when relatingto the complex, refers to any portion of the complex which can bespecifically bound by the antibody or antibody fragment, such that theantibody or antibody fragment is effectively incapable of specificallybinding: (i) the APM of the complex not bound to the antigen of thecomplex; (ii) an APM/antigen complex composed of the APM of the complexand an antigen other than that of the complex; or (iii) an APM/antigencomplex composed of an APM other than that of the complex and anyantigen restricted by such an APM, including the antigen of the complex.

As mentioned hereinabove, the antigen-presenting portion of the complexis typically a portion of the complex capable of specifically binding acognate TCR variable region. Antigen-presenting portions of complexeswhich can be specifically bound by the antibody or antibody fragmentcomprised in the composition-of-matter of the present invention aredescribed in further detail hereinbelow.

As used herein, the term peptide refers to a polypeptide composed of 50amino acid residues or less.

Depending on the application and purpose, the composition-of-matter maycomprise an antibody or an antibody fragment.

Preferably, the composition-of-matter comprises an antibody fragment.

Antibody fragments, various types of which are described in furtherdetail hereinbelow, have the advantage of generally being smaller thanan antibody while retaining essentially a substantially identicalbinding specificity of a whole antibody comprising the immunoglobulinvariable regions of the antibody fragment. Thus, a composition-of-matterof the present invention comprising an antibody fragment will begenerally smaller than one comprising an antibody, and will therebygenerally have superior biodistribution, and diffusion properties (forexample, systemically in-vivo, or in isolated tissues) than the latter.A smaller composition-of-matter will have the additional advantage ofbeing less likely to include moieties capable of causing sterichindrance inhibiting binding of the antibody or antibody fragmentcomprised in the composition-of-matter to the antigen-presenting portionof the complex. Also, the absence of some or all of an antibody constantregion (referred to herein as “constant region”), such as an Fc region,from a composition-of-matter of the present invention comprising anantibody fragment lacking such an Fc region will be advantageous forapplications involving exposure of the composition-of-matter to amolecule capable of specifically binding such a constant region and inwhich such binding is undesirable. Typically this may involve anundesired binding of an Fc region comprised in a composition-of-matterof the present invention exposed to a cognate Fc receptor, or anFc-binding complement component (for example, complement component C1q,present in serum). Fc receptors are displayed on the surface of numerousimmune cell types, including: professional APCs, such as dendriticcells; B lymphocytes; and granulocytes such as neutrophils, basophils,eosinophils, monocytes, macrophages, and mast cells. In particular, theabsence of a functional constant region, such as the Fc region, from thecomposition-of-matter will be particularly advantageous in applicationsin which the composition-of-matter is exposed to a specific ligand of aconstant region, such as a cognate Fc receptor or an Fc bindingcomplement component, capable of activating an undesired immuneresponse, such as an Fc receptor-mediated immune cell activation orcomplement component-mediated complement cascade, respectively, viainteraction with the constant region.

It will be appreciated by the ordinarily skilled artisan that in variouscontexts, the aforementioned Fc receptor-displaying cell types willfunction as APCs displaying/expressing the complex. Hence acomposition-of-matter of the present invention comprising an antibodyfragment lacking an Fc region may be advantageous for preventingundesired binding of the antibody or antibody fragment by Fc receptorsdisplayed by such cells, or for preventing consequent activation of suchcells.

Alternately, an antibody or antibody fragment of the present inventioncomprising such a functional constant region may be advantageous inapplications in which such an immune response is desirable. This will beparticularly desirable in applications involving use of thecomposition-of-matter to kill/damage target cells/tissues, as describedin further detail hereinbelow. A composition-of-matter of the presentinvention comprising an antibody or an antibody fragment including aconstant region, such as an Fc region, which may be convenientlyattached to a functional moiety will also be advantageous forapplications in which such attachment is desirable.

Furthermore, the use of a composition-of-matter of the present inventioncomprising an antibody fragment will be advantageous relative to oneemploying a whole antibody when employing recombinantly producing theantibody or antibody fragment due to antibody fragments being moreeconomical and efficient to synthesize due to their smaller sizerelative to whole antibodies.

Depending on the application and purpose, the composition-of-matter mayadvantageously comprise an antibody or antibody fragment having any ofvarious structural and/or functional characteristics. In particular,according to the teachings of the present invention, thecomposition-of-matter may advantageously comprise: (i) a monoclonal orpolyclonal antibody or antibody fragment; (ii) a monomeric or multimericform of antibody or antibody fragment; (iii) an antibody or antibodyfragment of any of various configurations or types (such as thosedescribed hereinbelow); (iv) an antibody or antibody fragment, orportion thereof, originating from any of various mammalian species; (v)an antibody or antibody fragment attached to any of various functionalmoieties; (vi) an antibody or antibody fragment capable of specificallybinding any of various particular complexes; and/or (vii) an antibody orantibody fragment capable of specifically binding the antigen-presentingportion of the complex with a desired affinity.

As mentioned hereinabove, depending on the application and purpose, theantibody or antibody fragment may be polyclonal or monoclonal.

As used herein, a composition-of-matter of the present inventioncomprising a “polyclonal” or “monoclonal” antibody or antibody fragmentis a population of molecules of the composition-of-matter comprising apolyclonal or monoclonal population of the antibody or antibodyfragment, respectively.

As used herein, a composition-of-matter of the present inventioncomprising a “polyclonal” or “monoclonal” antibody or antibody fragmentis a population of composition-of-matter molecules of the presentinvention each comprising a monoclonal antibody or antibody fragment ora population of same.

Methods of generating monoclonal or polyclonal antibodies or antibodyfragments are described hereinbelow.

Preferably, according to the teachings of the present invention, theantibody or antibody fragment is monoclonal.

For applications benefiting from optimal reproducibility,standardization, or precision, such as analytical applications, asdescribed in further detail hereinbelow, a composition-of-mattercomprising a monoclonal antibody or antibody fragment will generally besuperior to one comprising a polyclonal antibody or antibody fragmentdirected at the antigen-presenting portion of the same complex. Amonoclonal antibody or antibody fragment will be particularlyadvantageous in instances where the antibody or antibody fragment hasbeen characterized as having a desired binding affinity/specificity forthe antigen-presenting portion of the complex. A composition-of-matterof the present invention comprising such an antibody or antibodyfragment will thus be optimal for an application, as will generally bethe case, benefiting from a composition-of-matter comprising an antibodyor antibody fragment capable of binding the antigen-presenting portionof the complex with the highest affinity/specificity possible.

As is described and demonstrated in the Examples section below, acomposition-of-matter comprising a monoclonal antibody fragment can beused to optimally practice various aspects of the present invention,including applications involving specific detection of the complex, orkilling/damaging of target cells/tissues.

Alternately, for applications wherein a composition-of-matter capable ofbinding one or more complexes with a spectrum of, or with variousdistinct affinities/specificities is desirable, a composition-of-matterof the present invention comprising a polyclonal antibody or antibodyfragment will be advantageous. In any case, where no monoclonal antibodyor antibody fragment having a desired binding affinity/specificity forthe antigen-presenting portion of the complex is available, acomposition-of-matter comprising a polyclonal antibody or antibodyfragment will nevertheless often be adequate since the heterogeneity ofa polyclonal antibody or antibody fragment mixture will often includeone or more antibodies or antibody fragments having an adequate bindingaffinity/specificity for the antigen-presenting portion of the complex.

As mentioned hereinabove, depending on the application and purpose, theantibody fragment may be any of various configurations or types.

Suitable antibody fragments include a complementarity-determining region(CDR) of an immunoglobulin light chain (referred to herein as “lightchain”), a CDR of an immunoglobulin heavy chain (referred to herein as“heavy chain”), a variable region of a light chain, a variable region ofa heavy chain, a light chain, a heavy chain, an Fd fragment, an Fv, asingle chain Fv, an Fab, an Fab′, and an F(ab′)₂.

Antibody fragments among the aforementioned antibody fragments whichcomprise whole or essentially whole variable regions of both light andheavy chains are defined as follows: (i) Fv, a fragment of an antibodymolecule consisting of the light chain variable domain (V_(L)) and theheavy chain variable domain (V_(H)) expressed as two chains (typicallyobtained via genetic engineering of immunoglobulin genes); (ii) singlechain Fv (also referred to in the art as “scFv”), a single chainmolecule including the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker (a single-chain Fv is typically obtained via genetic engineeringof immunoglobulin genes and polypeptide linker-encoding DNA); (iii) Fab,a fragment of an antibody molecule containing essentially a monovalentantigen-binding portion of an antibody generally obtained by suitablytreating the antibody with the enzyme papain to yield the intact lightchain and the heavy chain Fd fragment (the Fd fragment consists of thevariable and C_(H)1 domains of the heavy chain); (iv) Fab′, a fragmentof an antibody molecule containing a monovalent antigen-binding portionof an antibody typically obtained by suitably treating the antibodymolecule with the enzyme pepsin, followed by reduction of the resultantF(ab′)₂ fragment (two Fab′ fragments are obtained per antibodymolecule); and (v) F(ab′)₂, a fragment of an antibody moleculecontaining a monovalent antigen-binding portion of an antibody moleculetypically obtained by suitably treating the antibody molecule with theenzyme pepsin (i.e., an F(ab′)₂ consists of two Fab's connected by apair of disulfide bonds).

Depending on the application and purpose, the antibody fragment ispreferably an Fab, or a single chain Fv.

As is described and illustrated in the Examples section which follows,and as described in further detail below, a composition-of-matter of thepresent invention comprising an Fab may be employed to effectivelypractice the present invention, in particular aspects thereof involvingusing the composition-of-matter to detect the antigen-presenting portionof the complex.

As is described and illustrated in the Examples section which follows,and as described in further detail below, a composition-of-matter of thepresent invention comprising a single chain Fv may be utilized toeffectively practice the present invention, in particular aspectsthereof involving utilizing the composition-of-matter to kill/damagetarget cells/tissues.

It will be appreciated by the ordinarily skilled artisan that, due to anFab′ being essentially similar in structure to an Fab, acomposition-of-matter of the present invention comprising an Fab′ may beemployed interchangeably with one comprising an Fab, where such Fab′ andFab comprise essentially the same heavy and light chain variableregions. For applications, as will usually be the case, benefiting froma composition-of-matter of the present invention comprising an antibodyfragment capable of binding the antigen-presenting portion of thecomplex with the highest possible affinity, a composition-of-matter ofthe present invention comprising an F(ab′)₂ may be advantageouslyemployed over one comprising a monovalent antibody fragment, such as anFab, an Fab′ or a single chain Fv, due to the divalent binding of anF(ab′)₂ to the antigen-presenting portion of the complex relative to themonovalent binding of such a monovalent antibody fragment.

As mentioned hereinabove, depending on the application and purpose, theantibody or antibody fragment may originate from any of variousmammalian species.

Preferably, the antibody or antibody fragment is of human origin.

An antibody or antibody fragment of human origin may be derived asdescribed further hereinbelow, or as described in the Examples sectionwhich follows.

A composition-of-matter of the present invention comprising an antibodyor antibody fragment of human origin will generally be preferable forapplications involving administration of the composition-of-matter to anindividual. For example, such an antibody or antibody fragment willgenerally tend to be better tolerated immunologically than one of nonhuman origin since non variable portions of non human antibodies willtend to trigger xenogeneic immune responses more potent than theallogeneic immune responses triggered by human antibodies which willtypically be allogeneic with the individual. It will be preferable tominimize such immune responses since these will tend to shorten thehalf-life, and hence the effectiveness, of the composition-of-matter inthe individual. Furthermore, such immune responses may be pathogenic tothe individual, for example by triggering harmful inflammatoryreactions.

As used herein, the term “individual”, refers to a human.

Alternately, an antibody or antibody fragment of human origin, or ahumanized antibody, will also be advantageous for applications in whicha functional physiological effect, for example an immune responseagainst a target cell, activated by a constant region of the antibody orantibody fragment in the individual is desired. Such applicationsparticularly include those in which the functional interaction between afunctional portion of the antibody or antibody fragment, such as an Fcregion, with a molecule such as an Fc receptor or an Fc-bindingcomplement component, is optimal when such a functional portion is,similarly to the Fc region, of human origin.

Depending on the application and purpose, a composition-of-matter of thepresent invention comprising an antibody or antibody fragment includinga constant region, or a portion thereof, of any of various isotypes maybe employed. Preferably, the isotype is selected so as to enable orinhibit a desired physiological effect, or to inhibit an undesiredspecific binding of the composition-of-matter via the constant region orportion thereof. For example, for inducing antibody-dependent cellmediated cytotoxicity (ADCC) by a natural killer (NK) cell, the isotypewill preferably be IgG; for inducing ADCC by a mast cell/basophil, theisotype will preferably be IgE; and for inducing ADCC by an eosinophil,the isotype will preferably be IgE or IgA. For inducing a complementcascade the composition-of-matter will preferably comprise an antibodyor antibody fragment comprising a constant region or portion thereofcapable of initiating the cascade. For example, the antibody or antibodyfragment may advantageously comprise a Cyt domain of IgG or CP domain ofIgM to trigger a C1q-mediated complement cascade.

Conversely, for avoiding an immune response, such as the aforementionedone, or for avoiding a specific binding via the constant region orportion thereof, the composition-of-matter will preferably not comprisea constant region, or a portion thereof, of the relevant isotype.

As mentioned hereinabove, depending on the application and purpose, theantibody or antibody fragment may be attached to any of variousfunctional moieties. An antibody or antibody fragment, such as that ofthe present invention, attached to a functional moiety may be referredto in the art as an “immunoconjugate”.

Preferably, the functional moiety is a detectable moiety or a toxin. Anantibody or antibody fragment attached to a toxin may be referred to inthe art as an immunotoxin.

As is described and demonstrated in further detail hereinbelow, adetectable moiety or a toxin may be particularly advantageously employedin applications of the present invention involving use of thecomposition-of-matter to detect the antigen-presenting portion of thecomplex, or to kill/damage target cells/tissues, respectively.

The composition-of-matter may comprise an antibody or antibody fragmentattached to any of numerous types of detectable moieties, depending onthe application and purpose.

For applications involving using the composition-of-matter to detect theantigen-presenting portion of the complex, the detectable moietyattached to the antibody or antibody fragment is preferably a reportermoiety enabling specific detection of the antigen-presenting portion ofthe complex bound by the antibody or antibody fragment of thecomposition-of-matter.

While various types of reporter moieties may be utilized to detect theantigen-presenting portion of the complex, depending on the applicationand purpose, the reporter moiety is preferably a fluorophore or anenzyme. Alternately, the reporter moiety may be a radioisotope, such as[125]iodine, as is described and illustrated in the Examples sectionbelow.

A fluorophore may be advantageously employed as a detection moietyenabling detection of the antigen-presenting portion of the complex viaany of numerous fluorescence detection methods. Depending on theapplication and purpose, such fluorescence detection methods include,but are not limited to, fluorescence activated flow cytometry (FACS),immunofluorescence confocal microscopy, fluorescence in-situhybridization (FISH), fluorescence resonance energy transfer (FRET), andthe like.

Various types of fluorophores, depending on the application and purpose,may be employed to detect the antigen-presenting portion of the complex.

Examples of suitable fluorophores include, but are not limited to,phycoerythrin, fluorescein isothiocyanate (FITC), Cy-chrome, rhodamine,green fluorescent protein (GFP), blue fluorescent protein (BFP), Texasred, and the like.

Preferably, the fluorophore is phycoerythrin.

As is described and illustrated in the Examples section below, acomposition-of-matter of the present invention comprising an antibody orantibody fragment attached to a fluorophore, such as phycoerythrin, canbe used to optimally detect the antigen-presenting portion of thecomplex using various immunofluorescence-based detection methods.

Ample guidance regarding fluorophore selection, methods of linkingfluorophores to various types of molecules, such as an antibody orantibody fragment of the present invention, and methods of using suchconjugates to detect molecules which are capable of being specificallybound by antibodies or antibody fragments comprised in suchimmunoconjugates is available in the literature of the art [for example,refer to: Richard P. Haugland, “Molecular Probes: Handbook ofFluorescent Probes and Research Chemicals 1992-1994”, 5th ed., MolecularProbes, Inc. (1994); U.S. Pat. No. 6,037,137 to Oncoimmunin Inc.;Hermanson, “Bioconjugate Techniques”, Academic Press New York, N.Y.(1995); Kay M. et al., 1995. Biochemistry 34:293; Stubbs et al., 1996.Biochemistry 35:937; Gakamsky D. et al., “Evaluating ReceptorStoichiometry by Fluorescence Resonance Energy Transfer,” in “Receptors:A Practical Approach,” 2nd ed., Stanford C. and Horton R. (eds.), OxfordUniversity Press, UK. (2001); U.S. Pat. No. 6,350,466 to Targesome,Inc.]. While various methodologies may be employed to detect theantigen-presenting portion of the complex using a fluorophore, suchdetection is preferably effected as described and demonstrated in theExamples section below.

Alternately, an enzyme may be advantageously utilized as the detectablemoiety to enable detection of the antigen-presenting portion of thecomplex via any of various enzyme-based detection methods. Examples ofsuch methods include, but are not limited to, enzyme linkedimmunosorbent assay (ELISA; for example, to detect theantigen-presenting portion of the complex in a solution), enzyme-linkedchemiluminescence assay (for example, to detect the complex in anelectrophoretically separated protein mixture), and enzyme-linkedimmunohistochemical assay (for example, to detect the complex in a fixedtissue).

Numerous types of enzymes may be employed to detect theantigen-presenting portion of the complex, depending on the applicationand purpose.

Examples of suitable enzymes include, but are not limited to,horseradish peroxidase (HPR), β-galactosidase, and alkaline phosphatase(AP).

Preferably, the enzyme is horseradish peroxidase.

As is described in the Examples section which follows, acomposition-of-matter of the present invention comprising an antibody orantibody fragment attached to an enzyme such as horseradish peroxidasecan be used to effectively detect the antigen-presenting portion of thecomplex, such as via ELISA, or enzyme-linked immunohistochemical assay.

Ample guidance for practicing such enzyme-based detection methods isprovided in the literature of the art (for example, refer to: KhatkhatayM I. and Desai M., 1999. J Immunoassay 20:151-83; Wisdom G B., 1994.Methods Mol. Biol. 32:433-40; Ishikawa E. et al., 1983. J Immunoassay4:209-327; Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208;Schuurs A H. and van Weemen B K., 1980. J Immunoassay 1:229-49). Whilevarious methodologies may be employed to detect the antigen-presentingportion of the complex using an enzyme, such detection is preferablyeffected as described in the Examples section below.

The functional moiety may be attached to the antibody or antibodyfragment in various ways, depending on the context, application andpurpose.

A polypeptidic functional moiety, in particular a polypeptidic toxin,may be advantageously attached to the antibody or antibody fragment viastandard recombinant techniques broadly practiced in the art (forExample, refer to Sambrook et al., infra, and associated references,listed in the Examples section which follows). While variousmethodologies may be employed, attaching a polypeptidic functionalmoiety to the antibody or antibody fragment is preferably effected asdescribed and illustrated in the Examples section below.

A functional moiety may also be attached to the antibody or antibodyfragment using standard chemical synthesis techniques widely practicedin the art [for example, refer to the extensive guidelines provided byThe American Chemical Society (for example at:http://www.chemistry.org/portal/Chemistry)]. One of ordinary skill inthe art, such as a chemist, will possess the required expertise forsuitably practicing such such chemical synthesis techniques.

Alternatively, a functional moiety may be attached to the antibody orantibody fragment by attaching an affinity tag-coupled antibody orantibody fragment of the present invention to the functional moietyconjugated to a specific ligand of the affinity tag.

Various types of affinity tags may be employed to attach the antibody orantibody fragment to the functional moiety.

Preferably, the affinity tag is a biotin molecule, more preferably astreptavidin molecule.

A biotin or streptavidin affinity tag can be used to optimally enableattachment of a streptavidin-conjugated or a biotin-conjugatedfunctional moiety, respectively, to the antibody or antibody fragmentdue to the capability of streptavidin and biotin to bind to each otherwith the highest non covalent binding affinity known to man (i.e., witha Kd of about 10⁻¹⁴ to 10⁻¹⁵). A biotin affinity tag may be highlyadvantageous for applications benefiting from, as will oftentimes be thecase, a composition-of-matter of the present invention comprising amultimeric form of the antibody or antibody fragment, which may beoptimally formed by conjugating multiple biotin-attached antibodies orantibody fragments of the present invention to a streptavidin molecule,as described in further detail below.

As used herein the term “about” refers to plus or minus 10 percent.

Various methods, widely practiced in the art, may be employed to attacha streptavidin or biotin molecule to a molecule such as the antibody orantibody fragment to a functional moiety.

For example, a biotin molecule may be advantageously attached to anantibody or antibody fragment of the present invention attached to arecognition sequence of a biotin protein ligase. Such a recognitionsequence is a specific polypeptide sequence serving as a specificbiotinylation substrate for the biotin protein ligase enzyme. Ampleguidance for biotinylating a target polypeptide such as an antibodyfragment using a recognition sequence of a biotin protein ligase, suchas the recognition sequence of the biotin protein ligase BirA, isprovided in the literature of the art (for example, refer to: Denkberg,G. et al., 2000. Eur. J. Immunol. 30:3522-3532). Preferably, suchbiotinylation of the antibody or antibody fragment is effected asdescribed and illustrated in the Examples section below.

Alternately, various widely practiced methods may be employed to attacha streptavidin molecule to an antibody fragment, such as a single chainFv (for example refer to Cloutier S M. et al., 2000. MolecularImmunology 37:1067-1077; Dubel S. et al., 1995. J Immunol Methods178:201; Huston J S. et al., 1991. Methods in Enzymology 203:46;Kipriyanov S M. et al., 1995. Hum Antibodies Hybridomas 6:93; KipriyanovS M. et al., 1996. Protein Engineering 9:203; Pearce L A. et al., 1997.Biochem Molec Biol Intl 42:1179-1188).

Functional moieties, such as fluorophores, conjugated to streptavidinare commercially available from essentially all major suppliers ofimmunofluorescence flow cytometry reagents (for example, Pharmingen orBecton-Dickinson). Standard recombinant DNA chemical techniques arepreferably employed to produce a fusion protein comprising streptavidinfused to a polypeptidic functional moiety. Standard chemical synthesistechniques may also be employed to form the streptavidin-functionalmoiety conjugate. Extensive literature is available providing guidancefor the expression, purification and uses of streptavidin orstreptavidin derived molecules (Wu S C. et al., 2002. Protein Expressionand Purification 24:348-356; Gallizia A. et al., 1998. ProteinExpression and Purification 14:192-196), fusion proteins comprisingstreptavidin or streptavidin derived molecules (Sano T. and Cantor C R.,2000. Methods Enzymol. 326:305-11), and modified streptavidin orstreptavidin derived molecules (see, for example: Sano T. et al., 1993.Journal of Biological Chemistry 270:28204-28209), including forstreptavidin or streptavidin derived molecules whose gene sequence hasbeen optimized for expression in E. coli (Thompson L D. and Weber P C.,1993. Gene 136:243-6).

The use of a composition-of-matter of the present invention comprisingan antibody or antibody fragment attached to a functional moiety forvarious purposes other than detection of the antigen-presenting portionof the complex, or killing/damaging target cells/tissues is alsoenvisaged by the present invention. In particular, acomposition-of-matter of the present invention comprising an antibody orantibody fragment attached to an affinity tag, or any substance,particle, virus or cell displaying/expressing such acomposition-of-matter, can be conveniently isolated or purified using anaffinity purification method employing as a capture ligand a specificligand of the affinity tag. Preferably, for such purposes, the affinitytag is a polyhistidine tag, and the purification method is effectedusing nickel as the specific ligand of the affinity tag.

A histidine tag is a peptide typically consisting of 4 to 8 histidineamino acid residues. Preferably a histidine tag composed of 6 histidineresidues, commonly referred to as a hexahistidine tag in the art, isemployed. Such histidine tags specifically bind nickel-containingsubstrates. Ample guidance regarding the use of histidine tags isavailable in the literature of the art (for example, refer to SheibaniN., 1999. Prep Biochem Biotechnol. 29:77). Purification of moleculescomprising histidine tags is routinely effected using nickel-basedaffinity purification techniques. An alternate suitable capture ligandfor histidine tags is the anti histidine tag single-chain antibody 3D5(Kaufmann, M. et al., 2002. J Mol Biol. 318, 135-47). While varioustechniques may be employed, purifying a composition-of-matter of thepresent invention comprising an antibody or antibody fragment attachedto a histidine tag is preferably effected as described and illustratedin the Examples section which follows.

The composition-of-matter may be purified using any of various suitablestandard and widely employed affinity chromatography techniques. Ampleguidance for practicing such techniques is provided in the literature ofthe art [for example, refer to: Wilchek M. and Chaiken I., 2000. MethodsMol Biol 147, 1-6; Jack G W., 1994. Mol Biotechnol 1, 59-86; Narayanan SR., 1994. Journal of Chromatography A 658, 237-258; Nisnevitch M. andFirer M A., 2001. J Biochem Biophys Methods 49, 467-80; Janson J C. andKristiansen T. in “Packings and Stationary Phases in ChromatographyTechniques”, Unger K K. (ed), Marcel Dekker, New York, pp. 747 (1990);Clonis Y D: HPLC of Macromolecules: A Practical Approach, IRL Press,Oxford, pp. 157 (1989); Nilsson J. et al., 1997. Protein Expr Purif.11:1-16].

Various affinity tags, other than those described hereinabove, may alsobe employed to attach the functional moiety to the antibody or antibodyfragment or to purify a composition-of-matter of the present inventioncomprising an antibody or antibody fragment attached to an affinity tag,or any substance, particle, virus or cell displaying/expressing such acomposition-of-matter.

Such affinity tags include, but are not limited to, a streptavidin tag(Strep-tag), an epitope tag (a moiety, usually peptidic, which can bespecifically bound with high affinity by a specific monoclonalantibody), a maltose-binding protein (MBP) tag, and a chitin-bindingdomain (CBD) tag.

Examples of epitope tags include an 11-mer Herpes simplex virusglycoprotein D peptide, and an 11-mer N-terminal bacteriophage t7peptide, being commercially known as HSVTag and t7Tag, respectively(Novagen, Madison, Wis., USA), and 10- or 9-amino acid c-myc orHemophilus influenza hemagglutinin (HA) peptides, which are recognizedby the variable regions of monoclonal antibodies 9E10 and 12Ca5,respectively.

A Strep-tag is a peptide having the capacity to specifically bindstreptavidin. Ample guidance regarding the use of Strep-tags is providedin the literature of the art (see, for example: Schmidt, T G M. andSkerra, A. 1993. Protein Eng. 6:109; Schmidt T G M. et al., 1996.Journal of Molecular Biology 255:753-766; Skerra A. and Schmidt T G M.,1999. Biomolecular Engineering 16:79-86; Sano T. and Cantor C R. 2000.Methods Enzymol. 326, 305-11; and Sano T. et al., 1998. Journal ofChromatography B 715:85-91).

A suitable maltose-binding domain tag is malE-encoded maltose-bindingprotein which has the capacity to specifically bind a substrateincluding amylose such as, for example, an amylose-based affinitypurification column. Ample guidance regarding the use of maltose-bindingprotein as an affinity tag is provided in the literature of the art(see, for example: Guan M. et al., 2002. Protein Expr Purif. 26:229-34;Cattoli F and Sarti G C, 2002. Biotechnol Prog. 18:94-100).

A suitable chitin-binding domain tag is B. circulans cbd-encoded chitinbinding domain which has the capacity to specifically bind chitin. Ampleguidance regarding the use of maltose-binding protein as an affinity tagis provided in the literature of the art (see, for example: Humphries HE et al., 2002. Protein Expr Purif. 26:243-8; and Chong S. et al., 1997.Gene 192:271-81).

Thus, the functional moiety may be attached to the antibody or antibodyfragment via any of the aforementioned various affinity tags, dependingon the application and purpose.

As mentioned hereinabove, the functional moiety attached to the antibodyor antibody fragment may be a toxin.

For applications of the composition-of-matter involving killing/damagingof target cells/tissues, the toxin is preferably capable ofkilling/damaging the target cells/tissues when conjugated thereto as aconsequence of specific binding of the antibody or antibody fragment tothe antigen-presenting portion of the complex.

Any of various toxins may be attached to the antibody or antibodyfragment, to thereby generate an immunotoxin suitable, for example, tokill/damage target cells/tissues using a composition-of-mattercomprising such an immunotoxin.

Preferably, the toxin is Pseudomonas exotoxin A, more preferably aportion thereof comprising the translocation domain and/or an ADPribosylation domain. Preferably, the portion comprising thetranslocation domain and/or an ADP ribosylation domain is the toxin PE38KDEL. Generation of an immunotoxin comprising PE38 KDEL as a toxinmoiety is preferably effected as described and illustrated in theExamples section below. Ample guidance for generating such animmunotoxin is provided in the literature of the art (for example, referto: Brinkmann U. et al., 1991. Proc. Natl. Acad. Sci. U.S.A. 88:8616-20;and Brinkmann U., 2000. In-vivo 14:21-7).

Other types of toxins which may be attached to the antibody or antibodyfragment, depending on the application and purpose, in particular tokill/damage a target cell, include, but are not limited to, variousbacterial toxins, plant toxins, chemotherapeutic agents, andradioisotopes, respectively. Examples of toxins commonly used togenerate immunotoxins include ricin and Pseudomonas exotoxin A-derivedPE40 toxin. Alternately, immunotoxins may be generated with toxins suchas diphtheria toxin, pertussis toxin, or cholera toxin.

Ample guidance for selecting, generating and using immunotoxins isprovided in the literature of the art (for example, refer to: Knechtle SJ. 2001, Philos Trans R Soc Lond B Biol Sci. 356:681-9; Hall W A., 2001.Methods Mol. Biol. 166:139-54; Brinkmann U., 2000. In-vivo 14:21-7;Haggerty H G. et al., 1999. Toxicol Pathol. 27:87-94; Chaplin J W.,1999. Med Hypotheses 52:133-46; Wu M., 1997. Br J. Cancer. 75:1347-55;Hall W A. 1996, Neurosurg Clin N Am. 7:537-46; Pasqualucci L. et al.,1995. Haematologica 80:546-56; Siegall C B., 1995. Semin Cancer Biol.6:289-95; Grossbard M L. et al., Clin Immunol Immunopathol. 76:107-14;Ghetie M A and Vitetta E S., 1994. Curr Opin Immunol. 6:707-14;Grossbard M L and Nadler L M., 1994. Semin Hematol. 31:88-97; Frankel AE., 1993. Oncology (Huntingt) 7:69-78; Pai L H. and Pastan I., 1993.JAMA. 269:78-81; Boon, T. and van der Bruggen, P., 1996. J. Exp. Med.183:725-729; Renkvist, N. et al., 2001. Cancer Immunol Immunother.50:3-15; Rosenberg, S. A., 2001. Nature 411:380-384; and U.S. Pat. No.5,677,274).

As mentioned hereinabove, depending on the application and purpose, thecomposition-of-matter may advantageously comprise a monomeric ormultimeric form of the antibody or antibody fragment.

A composition-of-matter of the present invention comprising a multimericform of the antibody or antibody fragment will generally bind theantigen-presenting portion of the complex with higher avidity, andthereby with higher affinity, than one comprising a monomeric form ofthe antibody or antibody fragment. Hence, a composition-of-matter of thepresent invention comprising a multimeric form of the antibody orantibody fragment may be advantageous for applications benefiting from,as will usually be the case, a reagent capable of specifically bindingthe antigen-presenting portion of the complex with the highest affinitypossible.

As is described and illustrated in the Examples section below, acomposition-of-matter of the present invention comprising a multimericform of an antibody or antibody fragment may be advantageously employedto effectively practice the method of the present invention, inparticular with respect to applications involving using thecomposition-of-matter to specifically detect the antigen-presentingportion of the complex.

Various methods may be employed to generate a composition-of-matter ofthe present invention comprising a multimeric form of the antibody orantibody fragment.

Preferably, the multimeric form of the antibody or antibody fragment isgenerated by binding a plurality of antibodies or antibody fragmentsattached to an affinity tag to a multimerizing molecule capable ofspecifically and simultaneously binding such a plurality of affinitytags. Alternately, the multimeric form of the antibody or antibodyfragment may be generated by attaching a plurality of antibodies orantibody fragments of the present invention to a moiety capable ofautomultimerizing, so as to thereby multimerize such a plurality ofantibodies or antibody fragments.

Any of various types of multimerizing molecule/affinity tag combinationsmay be employed to generate the multimeric form of the antibody orantibody fragment of the present invention.

Preferably, such a combination consists of a biotin affinity tag, and astreptavidin multimerizing molecule, which, as described hereinabove,bind to each other with the highest affinity known to man, and hencewill normally generate an optimally stable multimeric form of anantibody or antibody fragment of the present invention.

For certain applications a composition-of-matter of the presentinvention comprising a monomeric form of the antibody or antibodyfragment may be advantageous. Such a composition-of-matter, due to itsrelatively small size may be advantageous for applications, such asin-vivo applications, benefiting from optimal biodistribution and/ordiffusion thereof.

As is described and illustrated in the Examples section which follows, acomposition-of-matter of the present invention comprising a monomericform of an antibody or antibody fragment of the present invention may beadvantageously utilized, for example, in applications where such anantibody or antibody fragment is attached to a toxin to kill/damagetarget cells.

Preferably, the composition-of-matter comprises an antibody or antibodyfragment capable of specifically binding a complex in which the APM isan MHC class I molecule and the antigen is an MHC class I-restrictedantigen (referred to herein as “MHC class I/antigen complex”).

Alternately, the composition-of-matter may comprise an antibody orantibody fragment capable of specifically binding the antigen-presentingportion of a complex in which the APM is an MHC class II molecule andthe antigen is an MHC class II-restricted antigen (“MHC class II/antigencomplex”), or the APM is a CD1 molecule and the antigen is a CD1molecule and the antigen is a CD1-restricted antigen (“CD1/antigencomplex”). The composition-of-matter may also comprise an antibody orantibody fragment capable of specifically binding a complex structurallyand/or functionally analogous to an APM/antigen complex such as one ofthe aforementioned MHC- or CD1-based complexes.

The composition-of-matter may comprise an antibody or antibody fragmentcapable of specifically binding the antigen-presenting portion of any ofvarious particular MHC class II/antigen complexes. For example, theantigen-presenting portion of an MHC class II/antigen complex having asan APM an HLA-DP, HLA-DQ or HLA-DR molecule.

A composition-of-matter of the present invention may comprise anantibody or antibody fragment capable of specifically binding theantigen-presenting portion of a complex composed of an MHC class IImolecule and any of various MHC class II-restricted antigens, which aregenerally peptides about 10 to 30 amino acid residues in length. Suchpeptides generally have particular chemical compositions enabling theirspecific binding to a particular MHC class II molecule (for example,refer to: Fairchild P J., 1998. J Pept Sci. 4:182; Rammensee H G., 1995.Curr Opin Immunol. 7:85; Sinigaglia F. and Hammer J., 1994. APMIS.102:241; and Hobohm U. and Meyerhans A., 1993. Eur J. Immunol. 23:1271).

The composition-of-matter may comprise an antibody or antibody fragmentcapable of specifically binding the antigen-presenting portion of any ofvarious particular CD1/antigen complexes. For example, theantigen-presenting portion of a CD1/antigen complex having as an APM aCD11a, CD11b, CD11c or CD1d molecule.

A composition-of-matter of the present invention may comprise anantibody or antibody fragment capable of specifically binding theantigen-presenting portion of a complex composed of a CD1 molecule andany of various CD1-restricted antigens, which may be either peptides ormore typically lipids. For example: CD1b and CD1c molecules both havethe capacity to specifically associate with CD1b- or CD1-c-restrictedlipoarabinomannan, mycolic acid, or glucose monomycolate antigens; CD1chas the capacity to specifically associate with CD1c-restrictedpolyisoprenyl glycolipid antigens; and CD1d has the capacity tospecifically associate with CD1d-restricted glycophosphatidylinositol(GPI) anchor lipid and glycosylceramide lipid antigens.

The composition-of-matter may comprise an antibody or antibody fragmentcapable of specifically binding the antigen-presenting portion of any ofvarious particular MHC class I/antigen complexes, for example, an MHCclass I/antigen complex having as an MHC class I APM an HLA-A, HLA-B, orHLA-C molecule (referred to herein as “HLA-A/antigen complex”,“HLA-B/antigen complex”, or “HLA-A/antigen complex”, respectively).

While the composition-of-matter may comprise an antibody or antibodyfragment capable of specifically binding the antigen-presenting portionof a complex in which the APM is any of various HLA-A molecules, thecomposition-of-matter is preferably capable of binding theantigen-presenting portion of one in which the HLA-A molecule is HLA-A2.

As is described and illustrated in Examples section below, acomposition-of-matter of the present invention comprising an antibody orantibody fragment capable of specifically binding a complex having anHLA-A2 molecule as APM can be used to effectively practice variousembodiments of the present invention.

A composition-of-matter of the present invention may comprise anantibody or antibody fragment capable of specifically binding theantigen-presenting portion of a complex composed of an MHC class Imolecule and any of various MHC class I-restricted antigens, which aretypically peptides about 9 to 11 amino acid residues in length. Suchpeptides generally have particular chemical compositions enabling theirspecific binding to a particular MHC class I molecule (for example,refer to: Bianco A. et al., 1998. J Pept Sci. 4:471; Fairchild P J.,1998. J Pept Sci. 4:182; Falk K. and Rotzschke O., 1993. Semin Immunol.5:81; Rammensee H G., 1995. Curr Opin Immunol. 7:85; and Hobohm U. andMeyerhans A., 1993. Eur J. Immunol. 23:1271).

As described hereinabove, the composition-of-matter of the presentinvention comprises an antibody or antibody fragment capable ofspecifically binding the antigen-presenting portion of a particularcomplex composed of a human APM and an antigen derived from a pathogen.

While the composition-of-matter may comprise an antibody or antibodyfragment capable of specifically binding the antigen-presenting portionof a particular complex comprising an APM-restricted antigen derivedfrom essentially any type of pathogen, the pathogen is preferably anintracellular pathogen.

Alternately, the pathogen may a non-intracellular pathogen, such as abacterium, a fungus, a protozoan, a mycobacterium, a helminth, and thelike.

The composition-of-matter may comprise an antibody or antibody fragmentcapable of specifically binding the antigen-presenting portion of acomplex comprising an APM-restricted antigen derived from any of variousintracellular pathogens, including a virus, a mycobacterium, a bacterium(such as, for example, Listeria monocytogenes), and a protozoan (suchas, for example, Leishmania or Trypanosoma).

Preferably the antibody or antibody fragment is capable of specificallybinding the antigen-presenting portion of a complex comprising anAPM-restricted antigen derived from a viral pathogen.

Examples of such viral pathogens include retroviruses, circoviruses,parvoviruses, papovaviruses, adenoviruses, herpesviruses, iridoviruses,poxviruses, hepadnaviruses, picornaviruses, caliciviruses, togaviruses,flaviviruses, reoviruses, orthomyxoviruses, paramyxoviruses,rhabdoviruses, bunyaviruses, coronaviruses, arenaviruses, andfiloviruses.

While the composition-of-matter may comprise an antibody or antibodyfragment capable of specifically binding the antigen-presenting portionof a complex comprising as APM-restricted antigen an antigen derivedfrom any of various retroviruses, the retrovirus is preferably human Tlymphotropic virus-1 (HTLV-1; also referred to as human T-cell leukemiavirus in the art).

Alternately, the retrovirus may be, for example, HTLV-2, a humanimmunodeficiency virus (HIV) causing acquired immunodeficiency syndrome(AIDS) such as HIV-1 or HIV-2, or the like.

The composition-of-matter may comprise an antibody or antibody fragmentcapable of specifically binding the antigen-presenting portion of acomplex comprising any of various antigens derived from HTLV-1.

Preferably, a composition-of-matter of the present invention comprisingan antibody or antibody fragment capable of specifically binding theantigen-presenting portion of a complex comprising as APM-restrictedantigen derived from HTLV-1, an antigen derived from Tax protein.

The composition-of-matter may comprise an antibody or antibody fragmentcapable of specifically binding the antigen-presenting portion of acomplex comprising as APM-restricted antigen any of various Tax proteinderived antigens, and having an antigen binding region comprising any ofvarious amino acid sequences.

Preferably, the antibody or antibody fragment comprises an antibody orantibody fragment: (i) capable of specifically binding theantigen-presenting portion of a complex comprising as Tax proteinderived APM-restricted antigen a peptide comprising amino acid residues11 to 19 of Tax protein, a peptide having the amino acid sequence setforth in SEQ ID NO: 3, or preferably both; (ii) having anantigen-binding region including a maximal number of amino acidsequences corresponding to one selected from the group of amino acidsequences set forth in SEQ ID NOs: 14 to 97; or (iii) preferably both.

As is described and illustrated in the Examples section below, acomposition-of-matter of the present invention comprising an antibody orantibody fragment: (i) capable of specifically binding a complex havingas APM-restricted antigen a peptide comprising amino acid residues 11 to19 of Tax protein having the amino acid sequence set forth in SEQ ID NO:3; and (ii) having an antigen-binding region including amino acidsequences corresponding to those set forth in SEQ ID NOs: 14 to 97 canbe used to effectively practice various embodiments of the presentinvention, involving using the composition-of-matter for detecting theantigen-presenting portion of the complex, or killing targetcells/tissues.

It will be appreciated that a cell infected with a pathogen, and an APCexposed to the pathogen, or an antigen thereof, may express distinctcomplexes comprising different APMs and/or different antigens derivedfrom the pathogen, and that hence, the composition-of-matter may beadvantageously selected so as to selectively bind one or the other ofsuch cell types. This may be advantageously applied in numerousapplications of the present invention, such as, for example, when usingthe composition-of-matter, as described hereinbelow, to treat a diseaseassociated with a pathogen in an individual by selectivelykilling/damaging cells infected with the pathogen displaying oneparticular complex of an APM and an antigen derived from the pathogenwithout killing/damaging benign or beneficial APCs displaying adifferent complex of an APM and an antigen derived from the pathogen.

As mentioned hereinabove, depending on the application and purpose, theantibody or antibody fragment may be selected capable of binding theantigen-presenting portion of the complex with a desired affinity.

Preferably, the desired affinity is as high as possible. Acomposition-of-matter of the present invention comprising an antibody orantibody fragment having as high as possible a binding affinity for theantigen-presenting portion of the complex will generally enableoptimally stable conjugation of a functional moiety to theantigen-presenting portion of the complex, and thereby detection of theantigen-presenting portion of the complex with optimal sensitivity, orkilling/damaging of target cells/tissues with optimal efficiency.

Preferably, the affinity is characterized by a dissociation constant(K_(d)) selected from the range of 1×10⁻² molar to 5×10⁻³ molar, morepreferably 5×10⁻³ molar to 5×10⁻⁴ molar, more preferably 5×10⁻⁴ molar to5×10⁻⁵ molar, more preferably 5×10⁻⁵ molar to 5×10⁻⁶ molar, morepreferably 5×10⁻⁶ molar to 5×10⁻⁷ molar, more preferably 5×10⁻⁷ molar to5×10⁻⁸ molar, more preferably 5×10⁻⁸ molar to 5×10⁻⁹ molar, morepreferably 5×10⁻⁹ molar to 5×10⁻¹⁰ molar, more preferably 5×10⁻¹⁰ molarto 5×10⁻¹¹ molar, more preferably 5×10⁻¹¹ molar to 5×10⁻¹² molar, morepreferably 5×10⁻¹² molar to 5×10⁻¹³ molar, more preferably 5×10⁻¹³ molarto 5×10⁻¹⁴ molar, more preferably 5×10⁻¹⁴ molar to 5×10⁻¹⁵ molar, andmost preferably 5×10⁻¹⁵ molar to 5×10⁻¹⁶.

As is illustrated in the Examples section below, an antibody or antibodyfragment capable of binding the antigen-presenting portion of a complexwith an affinity characterized by a dissociation constant of about 10⁻⁹molar can be generated using the protocol set forth therein.

As is described and illustrated in the Examples section which follows, acomposition-of-matter of the present invention comprising an antibody orantibody fragment having a binding affinity for the antigen-presentingportion of the complex characterized by a dissociation constant of about10⁻⁹ molar can be used to effectively practice various embodiments ofthe present invention, including those involving using thecomposition-of-matter for detecting the antigen-presenting portion ofthe complex, or for killing/damaging target cells/tissues.

Various methods may be employed to obtain the antibody or antibodyfragment capable of specifically binding the antigen-presenting portionof the complex.

Preferably, the antibody or antibody fragment is obtained by screening acombinatorial antibody or antibody fragment display library for anelement of the library displaying an antibody or antibody fragmentcapable of binding the antigen-presenting portion of the complexconjugated to a substrate with the desired affinity. Preferably, wherethe antibody or antibody fragment is an Fab, this may be advantageouslyeffected by screening an Fab-phage library on substrate-immobilizedsingle-chain MHC/peptide complex, preferably as described in theExamples section below. Ample guidance for identifying an antibody orantibody fragment capable of specifically binding the complex isprovided in the literature of the art (for example, for generation of ahuman derived antibody or antibody fragment refer, for example, to:Chames, P. et al., 2000. Proc. Natl. Acad. Sci. U.S.A. 97:7969-7974;Denkberg, G. et al., 2002. Proc. Natl. Acad. Sci. U.S.A. 99:9421-9426;and Lev, A. et al., 2002. Cancer Res. 62:3184-3194; for generation of anon human derived antibody or antibody fragment refer, for example, to:Aharoni, R. et al., 1991. Nature 351:147-150; Andersen, P. S. et al.,1996. Proc. Natl. Acad. Sci. U.S.A 93:1820-1824; Dadaglio, G. et al.,1997. Immunity 6:727-738; Day, P. M. et al., 1997. Proc. Natl. Acad.Sci. U.S.A. 94:8064-8069; Krogsgaard, M. et al., 2000. J. Exp. Med.191:1395-1412; Murphy, D. B. et al., 1989. Nature 338:765-768; Porgador,A. et al., 1997. Immunity 6:715-726; Reiter, Y. et al., Proc. Natl.Acad. Sci. U.S.A. 94:4631-4636; Zhong, G. et al., 1997. Proc. Natl.Acad. Sci. U.S.A. 94:13856-13861; Zhong, G. et al., 1997. J. Exp. Med.186:673-682; Orlandi D. R. et al., 1989. Proc Natl Acad Sci USA.86:3833-3837; for a general reference, refer to Winter G. et al., 1991.Nature 349:293-299).

Further guidance for generating the antibody or antibody fragmentcomprised in the composition-of-matter of the present invention isprovided hereinbelow.

It will be appreciated by the ordinarily skilled artisan that generatingan antibody or antibody fragment of a desired affinity, for example onecharacterized by a dissociation constant as high as 10⁻¹² for a desiredantigenic determinant can be achieved using common art techniques.

The composition-of-matter may be used per se or it can be formulated asan active ingredient in a pharmaceutical composition.

Thus, as described hereinabove, the present invention provides, and maybe practiced, depending on the application and purpose using, acomposition-of-matter comprising: (i) a monoclonal or polyclonalantibody or antibody fragment; (ii) a monomeric or multimeric form of anantibody or antibody fragment; (iii) an antibody or antibody fragmentcharacterized by any of various configurations; (iv) an antibody orantibody fragment or a portion thereof derived from any of variousmammalian species; (v) an antibody or antibody fragment capable ofspecifically binding the antigen-presenting portion of any of variousspecific human APM/pathogen-derived antigen complexes; and/or (vi) anantibody or antibody fragment capable of specifically binding theantigen-presenting portion of a particular human APM/pathogen-derivedantigen complex with a desired affinity.

While further reducing the present invention to practice, geneticsequences encoding an antibody fragment of the present invention wereisolated.

Thus, according to another aspect of the present invention there isprovided an isolated polynucleotide comprising a nucleic acid sequenceencoding an antibody fragment of the present invention.

Depending on the application and purpose, the isolated polynucleotidepreferably further comprises a nucleic acid sequence encoding a coatprotein of a virus, a detectable moiety, and a toxin.

Preferably, in order to enable generation of a chimeric polypeptidecomprising the antibody fragment fused to the coat protein of the virus,the detectable moiety or the toxin, the nucleic acid sequence encodingthe polypeptide is translationally fused with that encoding the antibodyfragment. Nucleic acid sequences encoding polypeptides may betranslationally fused in a polynucleotide by cloning the structuralsequences of such nucleic acid sequences in-frame relative to each otherin the polynucleotide without intervening transcriptional/translationalstop codons, or any other sequences, present between such structuralsequences capable of preventing production of a chimeric polypeptidecomprising the polypeptides encoded by such structural sequences.

An antibody fragment attached to a coat protein of a virus can be usedto generate a virus displaying the antibody fragment by virtue of theantibody fragment being fused to the coat protein of the virus.Generating such a virus may be effected as described in further detailhereinbelow, and in the Examples section which follows.

While various methods may be used to generate the isolatedpolynucleotide, the isolated polynucleotide is preferably generated asdescribed in the Examples section, below.

As is described and illustrated in the Examples section below, anisolated polynucleotide of the present invention can be used to generatean antibody fragment or conjugate thereof with a coat protein of avirus, a detectable moiety, and/or a toxin suitable for generating thecomposition-of-matter of the present invention.

While reducing the present invention to practice nucleic acid constructscapable of expressing the polynucleotide of the present invention wereisolated or generated.

Thus, the present invention provides a nucleic acid construct comprisingthe isolated polynucleotide of the present invention and a promotersequence for directing transcription thereof in a host cell.

While various promoter sequences may be employed capable of directingtranscription of the isolated polynucleotide in various types of hostcell, depending on the application and purpose, the promoter sequence ispreferably capable of directing transcription thereof in a prokaryote.

The promoter sequence may be capable of directing transcription of thepolynucleotide in any of various suitable prokaryotes.

Preferably, the prokaryote is E. coli.

In order to enable regulatable transcription of the nucleic acidsequence in the host cell, the promoter sequence is preferably furthercapable of directing inducible transcription of the nucleic acidsequence in the host cell.

Various types of promoter sequences capable of directing transcriptionor inducible transcription of the polynucleotide in the host cell, suchas a suitable E. coli cell may be employed.

Preferably, the promoter sequence is a T7 promoter sequence.

It will be appreciated by the skilled artisan that a construct of thepresent invention comprising a T7 promoter sequence for directingtranscription of the polynucleotide can be used to efficiently induciblyexpress in a suitable E. coli host cell the antibody fragment of thepresent invention, or a conjugate thereof with a coat protein of avirus, detectable moiety, and/or toxin.

Preferably, the nucleic acid construct is isolated or assembled, and isused to inducibly produce the antibody fragment of the present inventionin a host cell as is described and demonstrated in the Examples sectionbelow.

As described hereinabove, the nucleic acid construct may be expressed invarious types of host cells. For example, the nucleic acid construct maybe advantageously expressed in a eukaryotic host cell, such as amammalian cell or a plant cell.

Methods of expressing nucleic acid constructs encoding antibodyfragments in eukaryotic cells are widely practiced by the ordinarilyskilled artisan.

Plant cells expressing the nucleic acid construct can be used togenerate plants expressing the nucleic acid construct, thereby enablinginexpensive and facile production of large quantities of antibody whichcan be harvested, processed and stored using existent infrastructure.

Expression of the nucleic acid construct of the present invention inplants can be used to produce plants expressing various forms of thecomposition-of-matter of the present invention, includingimmunoconjugates such as immunotoxins.

Ample guidance for expressing nucleic acid constructs encoding antibodyfragments, such as nucleic acid constructs encoding immunotoxins, inplant cells, and thereby in plants, is provided in the literature of theart (for example, refer to: Peeters K. et al., 2001. Vaccine 19:2756-61;De Jaeger G. et al., 2000. Plant Mol Biol. 43:419-28; Fischer R. et al.,2000. J Biol Regul Homeost Agents. 14:83-92; Fischer R. et al., 1999.Biotechnol Appl Biochem. 30:101-8; and Russell D A., 1999. Curr TopMicrobiol Immunol. 240:119-38).

While reducing the present invention to practice, viruses comprising thenucleic acid construct of the present invention, and a coat proteinfused to an antibody fragment of the present invention were isolated orgenerated.

Thus, the present invention provides a virus comprising the nucleic acidconstruct of the present invention and/or a coat protein fused to anantibody fragment of the present invention.

The virus of the present invention can be used in various applications,such as, for example, for selecting an antibody fragment of the presentinvention having a desired binding affinity/specificity for theantigen-presenting portion of the complex. Alternately, such a virus maybe used for propagating the antibody fragment or the nucleic acidconstruct. Preferably, such propagation is effected by using the virusto infect a host cell.

Any of various types of viruses comprising an antibody fragment of thepresent invention fused to any of various types of coat protein may beused.

Preferably, the virus is a filamentous phage and the coat protein ispIII.

While various methods may be employed to obtain and utilize the virus,it is preferably obtained and utilized as described and demonstrated inthe Examples section which follows.

While reducing the present invention to practice, host cells comprisingthe nucleic acid construct were generated and used to produce antibodyfragments of the present invention.

Thus, the present invention provides a host cell comprising the nucleicacid construct.

While the host cell may be advantageously used in various applications,it is preferably used to produce the antibody fragment, as mentionedhereinabove. Alternately, the host cell may be used to propagate thenucleic acid construct.

Various types of host cell may be used to practice the presentinvention, depending on the application and purpose.

Preferably, the host cell is a prokaryotic cell.

Alternately, the host cell may be a mammalian cell (please refer to theantibody/antibody fragment production guidelines herein for descriptionof suitable mammalian cells, and methods of their use).

While any of various types of prokaryotic host cells may be utilized,the prokaryotic cell is preferably an E. coli cell.

While various methods may be employed to obtain and utilize the E. colihost cell, for example, to produce the antibody or antibody fragment, itis preferably obtained and utilized as described and demonstrated in theExamples section which follows.

While reducing the present invention to practice the capacity of thecomposition-of-matter to enable specific detection of theantigen-presenting portion of a particular human APM/pathogen-derivedantigen complex was demonstrated.

Thus, according to another aspect of the present invention there isprovided a method of detecting the antigen-presenting portion of thecomplex.

The method is effected by exposing the antigen-presenting portion of thecomplex to a composition-of-matter of the present invention to therebyobtain a conjugate of the antigen-presenting portion of the complex andthe antibody or antibody fragment comprised in thecomposition-of-matter. Once the conjugate is formed, the method furthercomprises detecting the antibody or antibody fragment of the conjugateso as to thereby detect the antigen-presenting portion of the complex.

The method according to this aspect of the present invention can be usedto detect the antigen-presenting portion of the complex in any ofvarious contexts and applications.

In particular, as described hereinbelow, the method can be used todiagnose an infection by a pathogen in an individual.

Depending on the application and purpose, various methods may beutilized to expose the antigen-presenting portion of the complex to thecomposition-of-matter, according to the teachings of the presentinvention.

When using the method for detecting the antigen-presenting portion of acomplex displayed/expressed by target cells/tissues, or of a compleximmobilized on a surface, the antigen-presenting portion of the complexis preferably exposed to the composition-of-matter by exposing thetarget cells/tissues, or the surface-immobilized antigen-presentingportion of the complex, respectively, to the composition-of-matter.

For certain applications, the biological sample may be advantageouslyobtained from an individual prior to contacting thecomposition-of-matter with the biological sample. Alternately, thecomposition-of-matter may be contacted with the biological sample byadministering the composition-of-matter to the individual.

As described hereinabove, once the composition-of-matter and theantigen-presenting portion of the complex exposed to thecomposition-of-matter form the conjugate, the method further comprisesdetecting the antibody or antibody fragment of the conjugate so as tothereby detect the antigen-presenting portion of the complex.

While various methods may be employed to detect the antibody or antibodyfragment of the antigen-presenting portion of the complex, theantigen-presenting portion of the complex is preferably detected byusing a composition-of-matter of the present invention comprising anantibody or antibody fragment attached a detectable moiety, anddetecting the antibody or antibody fragment by detecting the detectablemoiety attached thereto.

As described hereinabove, various detectable moieties may be used todetect the antigen-presenting portion of the complex in the context ofvarious detection assays, depending on the application and purpose.

Preferably, the method according to this aspect of the present inventionis used to detect the antigen-presenting portion of a complex in abiological sample. Alternately, the method may be used to detect theantigen-presenting portion of a complex immobilized on a non-cellularsurface, such as an the surface of an ELISA plate.

While the method may be used to detect the antigen-presenting portion ofthe complex in essentially any type of biological sample, it ispreferably applied to detect the antigen-presenting portion of a complexdisplayed/expressed by target cells/tissues.

Preferably, the target cells are pathogen infected cells displaying thecomplex, or APCs displaying the complex, such as professional APCs,dendritic cells, B lymphocytes, granulocytes, neutrophils, basophils,eosinophils, monocytes, macrophages, and mast cells.

It will be appreciated that since, as described hereinabove, thecomposition-of-matter may comprise an antibody or antibody fragmentcapable of specifically binding a complex comprising as APM-restrictedantigen an antigen derived from essentially any pathogen, the methodaccording to this aspect of the present invention can be used to detecta complex comprising as APM-restricted antigen, an antigen derived fromessentially any pathogen.

Preferably, the method is used to detect target cellsdisplaying/expressing a particular complex comprising as APM-restrictedantigen, an HTLV-1 derived antigen.

Preferably, the method according to this aspect of the present inventionis effected as described in the Examples section which follows.

As is demonstrated in the Examples section below, practicing the methodaccording to the protocol set forth therein can be used in numerouscontexts to detect with optimal specificity and sensitivity cellsdisplaying a particular complex comprising as APM-restricted antigen, anHTLV-1 derived antigen, or such a complex immobilized on a non cellularsurface.

Thus, the method according to this aspect of the present invention maybe used to effectively and potently diagnose an infection by a pathogenin an individual.

It will be appreciated that since, as described hereinabove, this aspectof method of the present invention can be used to detect essentially anycomplex in essentially any context with optimal specificity and/orsensitivity, the method according to this aspect of the presentinvention can be used to optimally diagnose and characterize essentiallyany infection associated with essentially any pathogen.

For example, as described in the Examples section below, the methodaccording to this aspect of the present invention can be used tooptimally detect an APM/retrovirus-derived antigen. Thus, the method canbe used to optimally detect in an individual an infection by aretrovirus. Retrovirus are associated with a wide variety of diseasesincluding an array of malignancies, immunodeficiencies (notably AIDS),and neurological disorders, and syndromes as seemingly diverse asarthritis, osteopetrosis, and anemia. Thus, the method according to thisaspect of the present invention can be used, for example, to optimallydiagnose essentially all such diseases in an individual.

Preferably, the method according to this aspect of the present inventionis used to diagnose an HTLV-1 infection in an individual, since, asdescribed and demonstrated in the Examples section which follows, themethod according to this aspect of the present invention can be used todetect with optimal sensitivity and specificity a target cell displayinga complex comprising as APM-restricted antigen, an HTLV-1 derivedantigen. Diseases associated with HTLV-1 infection which may diagnosedand characterized using this according to this aspect of the presentinvention include adult T lymphocyte leukemia/lymphoma (ATLL; Yoshida M.et al., 1982. Proc Natl Acad Sci USA. 79:2031-2035), HTLV-I associatedmyelopathy/tropical virus spastic paraparesis (HAM/TSP; Osame M. et al.,1986. Lancet 1:1031-1032), Sjogren's syndrome, inflammatoryarthropathies, polymyositis, and pneumopathies (Coscoy L. et al., 1998.Virology 248: 332-341).

While reducing the present invention to practice, the capacity of thecomposition-of-matter of the present invention to enablekilling/damaging of target cells was demonstrated.

Thus, according to a further aspect of the present invention there isprovided a method of killing or damaging target cells.

According to the teachings of the present invention, the method iseffected by exposing the target cells to the composition-of-matter ofthe present invention.

The method may be effected so as to kill various types of target cellsin various ways, depending on the application and purpose.

Preferably, the method is effected by exposing target cells to acomposition-of-matter of the present invention comprising an antibody orantibody fragment attached to a toxin, so as to thereby kill/damage thetarget cells via the toxin.

Alternately, in an in-vivo context or an in-vitro equivalent thereof,the method may be effected by exposing target cells to acomposition-of-matter of the present invention comprising an antibody orantibody fragment including an Fc region, or portion thereof, capable ofspecifically binding a molecule capable of initiating an immuneresponse, such as a complement cascade or ADCC, directed against targetcells bound by such an antibody or antibody fragment, as describedhereinabove.

While the method according to this aspect of the present invention canbe used for killing/damaging target cells in any of various contexts andapplications, it is preferably employed to kill/damage target cells soas to treat a disease associated with a pathogen in an individual.

It will be appreciated that the method may also be used to kill/damagetarget cells in-vitro or in-vivo in an animal model, in particular totest and/or optimize killing/damaging of target cells using thecomposition-of-matter. Such testing and/or optimizing killing/damagingof target cells using the composition-of-matter may be advantageouslyapplied towards optimizing treatment of the disease in the individualusing the composition-of-matter.

When using the method according to this aspect of the present inventionfor optimizing use of the composition-of-matter to kill/damage targetcells for treating the disease, the method may be advantageouslyeffected by obtaining the target cells from the individual. One ofordinary skill in the art, such as a physician, will possess thenecessary expertise to obtain target cells from an individual.

Various types of target cells may be obtained from the individual foroptimizing use of the composition-of-matter to kill/damage target cells.Preferably, such target cells are cells infected with the pathogen sincesuch cells will be of particular utility for optimizing killing oftarget cells infected with the pathogen, and hence for optimizingtreatment of the disease in the individual.

It will be appreciated that since, as described hereinabove, thecomposition-of-matter may comprise an antibody or antibody fragmentcapable of binding with optimal specificity and affinity a complexcomprising as APM-restricted antigen an antigen derived from essentiallyany pathogen, the method according to this aspect of the presentinvention can be used to kill/damage cells displaying/expressing acomplex comprising as APM-restricted antigen, an antigen derived fromessentially any pathogen with optimal efficiency and specificity.

Preferably, the method is used to kill/damage target cellsdisplaying/expressing a particular complex comprising as APM-restrictedantigen, an HTLV-1 derived antigen.

Preferably, the method according to this aspect of the present inventionis effected as described in the Examples section which follows.

As is demonstrated in the Examples section below, practicing the methodaccording to the protocol set forth therein can be used to kill withoptimal efficiency and specificity cells displaying a particular complexcomprising as APM-restricted antigen, an HTLV-1 derived antigen.

Thus, the present invention provides a method of treating a diseaseassociated with a pathogen in an individual.

The method is effected by administering to the individual atherapeutically effective amount of a pharmaceutical compositioncomprising as an active ingredient a composition-of-matter of thepresent invention comprising as APM-derived antigen, an antigen derivedfrom the pathogen.

As described in detail hereinbelow, the pharmaceutical composition maybe administered in various ways.

As described hereinabove, the method according to this aspect of thepresent invention is preferably effected using a composition-of-mattercomprising an immunotoxin.

Ample guidance for treating a disease using an immunotoxin is providedin the literature of the art (for example, for general references referto: Knechtle S J. 2001, Philos Trans R Soc Lond B Biol Sci. 356:681-93;Kreitman R J., 2001. Methods Mol. Biol. 166:111-23; Brinkmann U., 2000.in-vivo 14:21-7; Ghetie M A and Vitetta E S., 1994. Curr Opin Immunol.6:707-14; Wu M., 1997. Br J. Cancer. 75:1347-55; Hall W A. 1996,Neurosurg Clin N Am. 7:537-46; Boon, T. and van der Bruggen, P., 1996.J. Exp. Med. 183:725-729; Renkvist, N. et al., 2001. Cancer ImmunolImmunother. 50:3-15; Rosenberg, S. A., 2001. Nature 411:380-384; andU.S. Pat. No. 5,677,274; for treatment of acquired immunodeficiencysyndrome (AIDS), refer, for example, to Chaplin J W., 1999. MedHypotheses 52:133-46; for treatment of brain tumors, refer, for example,to Hall W A., 2001. Methods Mol. Biol. 166:139-54; for treatment ofhaematological malignancies, refer, for example to: Pasqualucci L. etal., 1995. Haematologica 80:546-56; Grossbard M L. et al., Clin ImmunolImmunopathol. 76:107-14; and Grossbard M L and Nadler L M., 1994. SeminHematol. 31:88-97; for treatment of carcinomas, refer, for example, toSiegall C B., 1995. Semin Cancer Biol. 6:289-95; for treatment ofcancer, refer, for example, to: Frankel A E., 1993. Oncology (Huntingt)7:69-78; and Pai L H. and Pastan I., 1993. JAMA. 269:78-81).

The method can be used to treat various types of diseases associatedwith a pathogen using various methodologies taught by the presentinvention.

Preferably, the method is used to treat a disease associated with apathogen by killing/damaging pathogen infected cells. This may beadvantageously performed where the pathogenesis of the disease derivespredominantly from the pathogen infected cells.

Alternately, the method may be used to treat the disease, where thedisease involves a pathogenic immune response directed againstpathogen-infected cells by pathogenic T lymphocytes activated bypathogenic APCs displaying/expressing a complex comprising asAPM-restricted antigen, an antigen derived from the pathogen. This ispreferably effected, as described hereinabove, by using thecomposition-of-matter to kill/damage such pathogenic APCs. Alternately,the pathogenic immune response mediated by such pathogenic APCs may beinhibited as described hereinabove, by using a composition-of-matter ofthe present invention comprising an antibody or antibody fragmentcapable of specifically binding the pathogenic complex so as to therebyblock activation of the pathogenic T lymphocytes via engagement of theTCRs thereof by the complex.

It will be appreciated that since, as described hereinabove, this aspectof method of the present invention can be used to kill with optimalefficiency and specificity cells displaying/expressing essentially anyparticular complex, the method according to this aspect of the presentinvention can be used to optimally treat essentially any infectionassociated with essentially any pathogen in an individual.

For example, as described in the Examples section below, the methodaccording to this aspect of the present invention can be used tokill/damage with optimal efficiency and specificity cells displaying acomplex comprising as APM-restricted antigen, an antigen derived from aretrovirus. Thus, the method can be used to optimally treat, forexample, an infection associated with a retrovirus in an individual.

Thus, the method according to this aspect of the present invention canbe used, for example, to optimally treat the broad range of diseasesassociated with a retroviral infection described hereinabove.

Preferably, the method according to this aspect of the present inventionis used to treat an HTLV-1 infection in an individual, since, asdescribed and demonstrated in the Examples section which follows, themethod according to this aspect of the present invention can be used tokill with optimal efficiency and specificity target cells displaying acomplex comprising as APM-restricted antigen, an HTLV-1 derived antigen.

As described hereinabove, the antibody or antibody fragment of thepresent invention may be generated in numerous ways.

A monoclonal or polyclonal antibody or antibody fragment of the presentinvention may be generated via methods employing induction of in-vivoproduction of antibody or antibody fragment molecules, or culturing ofantibody or antibody fragment-producing cell lines. Ample guidance forpracticing such methods is provided in the literature of the art [forexample, refer to Harlow and Lane, “Antibodies: A Laboratory Manual”,Cold Spring Harbor Laboratory, New York, (1988)].

Cell culture-based methods of generating antibodies include thehybridoma technique, the human B-cell hybridoma technique, and theEpstein-Barr virus (EBV)-hybridoma technique (Kohler G. et al., 1975.Nature 256:495-497; Kozbor D. et al., 1985. J. Immunol. Methods81:31-42; Cote R J. et al., 1983. Proc Natl Acad Sci USA. 80:2026-2030;Cole S P. et al., 1984. Mol. Cell. Biol. 62:109-120).

Generating an antibody or antibody or antibody fragment of the presentinvention in-vivo may be advantageously effected by repeated injectionof a target antigen (e.g., one comprising the antigen-presenting portionof the complex) into a mammal in the presence of adjuvants according toa schedule which boosts production of antibodies in the serum. In caseswherein the target antigen is too small to elicit an adequateimmunogenic response (referred to as a “hapten” in the art), the haptencan be coupled to an antigenically neutral carrier such as keyholelimpet hemocyanin (KLH) or serum albumin [e.g., bovine serum albumin(BSA)] carriers (for example, refer to: U.S. Pat. Nos. 5,189,178 and5,239,078). Coupling a hapten to a carrier can be effected using variousmethods well known in the art. For example, direct coupling to aminogroups can be effected and optionally followed by reduction of the iminolinkage formed. Alternatively, the carrier can be coupled usingcondensing agents such as dicyclohexyl carbodiimide or othercarbodiimide dehydrating agents. Linker compounds can also be used toeffect the coupling; both homobifunctional and heterobifunctionallinkers are available from Pierce Chemical Company, Rockford, Ill. Theresulting immunogenic complex can then be injected into suitablemammalian subjects such as mice, rabbits, and the like. Followingin-vivo generation of an antibody, its serum titer in the host mammalcan readily be measured using immunoassay procedures which are wellknown in the art. Such a polyclonal antibody containing anti-serum maybe utilized as such, following purification thereof to generate a purepolyclonal or monoclonal antibody preparation. Such an anti-serum orpurified antibody preparation may also be modified in various ways,depending on the application and purpose, prior to use. Geneticsequences encoding an antibody isolated from such an anti-serum may bedetermined using standard art techniques, and used to recombinantlyproduce the antibody or a modification thereof, such as an antibodyfragment.

An antibody fragment of the present invention can be obtained usingvarious methods well known in the art. For example, such an antibodyfragment can be prepared by proteolytic hydrolysis of a parentalantibody or by recombinant expression in E. coli or mammalian cells(e.g., Chinese hamster ovary cell culture or other protein expressionsystems) of DNA encoding the fragment.

An F(ab′)₂ antibody fragment can be produced by enzymatic cleavage of aparental antibody with pepsin to provide a 5S fragment. This fragmentcan be further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages to produce a 3.5S monovalent Fab′ antibody fragment.

Enzymatic cleavage of a parental antibody with pepsin can be used todirectly produce two monovalent Fab′ fragments and an Fc fragment. Ampleguidance for practicing such methods is provided in the literature ofthe art (for example, refer to: Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647; Porter R R., 1959. Biochem J. 73:119-126).

As described hereinabove, an Fv is composed of paired heavy chainvariable and light chain variable domains. This association may benoncovalent (for example, refer to Inbar et al., 1972. Proc. Natl. Acad.Sci. U.S.A. 69:2659-62). Alternatively, as described hereinabove thevariable domains can be linked to generate a single chain Fv by anintermolecular disulfide bond, or such chains may be covalentlycross-linked using chemicals such as glutaraldehyde. A single chain Fvmay advantageously prepared by constructing a structural gene comprisingDNA sequences encoding the heavy chain variable domain and the lightchain variable domain connected by an oligonucleotide encoding a peptidelinker. The structural gene is inserted into an expression vector, whichis subsequently introduced into a host cell such as E. coli which willthen synthesize such a single chain Fv. Ample guidance for practicingsuch methods of producing a single chain Fv is provided in theliterature of the art (for example, refer to: Whitlow and Filpula, 1991.Methods 2:97-105; Bird et al., 1988. Science 242:423-426; Pack et al.,1993. Bio/Technology 11:1271-77; and Ladner et al., U.S. Pat. No.4,946,778).

Other methods of cleaving an antibody, such as separation of heavychains to form monovalent light-heavy chain fragments, further cleavageof fragments, or other enzymatic, chemical, or genetic techniques mayalso be used, so long as the fragment bind to the target antigen that isrecognized by the intact antibody.

A polypeptide comprising a complementarity determining region (CDR)peptide of an antibody can be obtained via recombinant techniques usinggenetic sequences encoding such a CDR, for example, by RT-PCR of mRNA ofan antibody-producing cell. Ample guidance for practicing such methodsis provided in the literature of the art (for example, refer to Larrickand Fry, 1991. Methods 2:106-10).

It will be appreciated that for human therapy or diagnostics, ahumanized antibody or antibody fragment may be advantageously used.Humanized non human (e.g., murine) antibodies are genetically engineeredchimeric antibodies or antibody fragments having—preferablyminimal—portions derived from non human antibodies. Humanized antibodiesinclude antibodies in which complementary determining regions of a humanantibody (recipient antibody) are replaced by residues from acomplementarity determining region of a non human species (donorantibody) such as mouse, rat or rabbit having the desired functionality.In some instances, Fv framework residues of the human antibody arereplaced by corresponding non human residues. Humanized antibodies mayalso comprise residues which are found neither in the recipient antibodynor in the imported complementarity determining region or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the complementarity determiningregions correspond to those of a non human antibody and all, orsubstantially all, of the framework regions correspond to those of arelevant human consensus sequence. Humanized antibodies optimally alsoinclude at least a portion of an antibody constant region, such as an Fcregion, typically derived from a human antibody (see, for example, Joneset al., 1986. Nature 321:522-525; Riechmann et al., 1988. Nature332:323-329; and Presta, 1992. Curr. Op. Struct. Biol. 2:593-596).Methods for humanizing non human antibodies or antibody fragments arewell known in the art. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non human.These non human amino acid residues are often referred to as importedresidues which are typically taken from an imported variable domain.Humanization can be essentially performed as described (see, forexample: Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988.Nature 332:323-327; Verhoeyen et al., 1988. Science 239:1534-1536; U.S.Pat. No. 4,816,567) by substituting human complementarity determiningregions with corresponding rodent complementarity determining regions.Accordingly, such humanized antibodies are chimeric antibodies, whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non human species. Inpractice, humanized antibodies may be typically human antibodies inwhich some complementarity determining region residues and possibly someframework residues are substituted by residues from analogous sites inrodent antibodies. Human antibodies or antibody fragments can also beproduced using various techniques known in the art, including phagedisplay libraries [see, for example, Hoogenboom and Winter, 1991. J.Mol. Biol. 227:381; Marks et al., 1991. J. Mol. Biol. 222:581; Cole etal., “Monoclonal Antibodies and Cancer Therapy”, Alan R. Liss, pp. 77(1985); Boerner et al., 1991. J. Immunol. 147:86-95). Humanizedantibodies can also be made by introducing sequences encoding humanimmunoglobulin loci into transgenic animals, e.g., into mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon antigenic challenge, human antibody production isobserved in such animals which closely resembles that seen in humans inall respects, including gene rearrangement, chain assembly, and antibodyrepertoire. Ample guidance for practicing such an approach is providedin the literature of the art (for example, refer to: U.S. Pat. Nos.5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016;Marks et al., 1992. Bio/Technology 10:779-783; Lonberg et al, 1994.Nature 368:856-859; Morrison, 1994. Nature 368:812-13; Fishwild et al.,1996. Nature Biotechnology 14:845-51; Neuberger, 1996. NatureBiotechnology 14:826; Lonberg and Huszar, 1995. Intern. Rev. Immunol.13:65-93).

Once an antibody or antibody or antibody fragment is obtained, it may beadvantageously tested for specific binding to the antigen-presentingportion of the complex, for example via ELISA, using surface-immobilizedtarget complex, as described in further detail hereinbelow, and in theExamples section which follows. Following confirmation of specificbinding of the antibody or antibody fragment to the antigen-presentingportion of the complex, various methods may be employed to modify theantibody or antibody fragment to display the desired binding affinityfor the antigen-presenting portion of the complex. Such methods includethose based on affinity maturation (for example, refer to: Chowdhury, P.S., and Pastan, I., 1999. Nat. Biotechnol. 17:568-72).

As described hereinabove, the present invention can be used to treat adisease associated with an infection by a pathogen in an individual byadministering a pharmaceutical composition comprising as an activeingredient a composition-of-matter of the present invention.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of active ingredients to an organism.

Herein the term “active ingredients” refers to the composition-of-matteraccountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered active ingredients. An adjuvant isincluded under these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular, intravenous,inrtaperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active ingredients withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethylcellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Alternately, for oral administration, the pharmaceutical composition maycomprise an edible part of a plant containing, for example theimmunotoxin of the present invention, as described hereinabove. Hence anindividual may consume such an immunotoxin in the form of a plant foodendogenously expressing the immunotoxin.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the active ingredients and a suitable powder base such as lactose orstarch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredients may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (nucleic acid construct) effective to prevent,alleviate or ameliorate symptoms of a disorder (e.g., ischemia) orprolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredients sufficient to exert adesired therapeutic effect (minimal effective concentration, MEC). TheMEC will vary for each preparation, but can be estimated from in vitrodata. Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Detection assays can beused to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredients. Thepack may, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as if further detailed above.

It is expected that during the life of this patent many relevant medicaldiagnostic techniques will be developed and the scope of the phrase“method of detecting” is intended to include all such new technologies apriori.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below.

Example 1 Generation of Reagents Capable of Binding with OptimalAffinity and Specificity Particular Human APM/Pathogen-Derived AntigenComplexes Applicable Towards Optimal Diagnosis, Characterization, andTreatment of Human Pathogen Infections

Diseases associated with a pathogen infection, such as a viralinfection, include numerous debilitating or lethal diseases of majormedical and economic impact, including influenza, the common cold, andacquired immunodeficiency syndrome (AIDS). One theoretically potentapproach which has been proposed for diagnosing, characterizing, andtreating such pathogen mediated diseases involves using compoundscapable of binding specific human antigen-presenting molecule(APM)/pathogen-derived peptide complexes. Such compounds could be usedto identify and characterize pathogen infected cells/tissues, or APCsexposed to viral antigens with optimal specificity, to deliver cytotoxicagents with optimal selectivity and efficiency to pathogen infectedcells, and to serve as uniquely potent tools for studying pathogenmediated pathogenesis involving viral antigen presentation. However, allprior art approaches of generating compounds capable of specificallybinding such complexes have failed to provide compounds capable ofbinding with optimal affinity/specificity human APM/pathogen-derivedantigen complexes. While reducing the present invention to practice, thepresent inventors have unexpectedly uncovered such compounds, asfollows.

Materials and Methods:

Cell Lines and Antibodies:

RMA-S-HHD is a TAP2 deficient cell line which expressesHLA-A2.1/Db-β₂-microglobulin single chain (Pascolo, S. et al., 1997. J.Exp. Med. 185:2043-2051). JY is a TAP and HLA-A2 positive EBVtransformed B lymphoblast cell line. APD is an HLA-A2 negative/HLA-A1positive B cell line. HUT 102 and RSCD4 are HLA-A2 negative andpositive, HTLV-1 infected human CD4 positive T lymphocyte cell lines,respectively.

G2D12 is an anti HLA-A2/G9-154 complex Fab used as a negative control(peptide G9-154 is derived from the melanoma specific gp100 protein).Monoclonal antibodies w6/32 and BB7.2 specifically bind correctlyfolded, peptide bound HLA (pan HLA), and HLA-A2, respectively.

Production of Biotinylated Soluble HLA-A2/Tax₁₁₋₁₉ Complex:

Soluble biotinylated HLA-A2/Tax₁₁₋₁₉ complex was generated as previouslydescribed (Denkberg, G. et al., 2000. Eur. J. Immunol. 30:3522-3532).Briefly, a construct was assembled for expression of single chain MHCfusion protein containing HLA-A2 fused to β₂-microglobulin, the BirArecognition sequence for site specific biotinylation at the C-terminus,and a hexahistidine tag fused to the C_(H)1 domain of the Fd chain. Inthis single chain fusion protein, HLA-A2 and β₂-microglobulin are fusedvia a flexible peptide linker. For expression of this single chain E.coli, transformants were generated using the construct, inclusion bodiescontaining the fusion protein were isolated from the periplasmicfraction of transformants by nickel affinity chromatography, and thefusion protein from inclusion bodies was refolded in-vitro in thepresence of a 5 to 10 fold molar excess of HLA-A2 restricted peptide soas to generate soluble, correctly folded and assembled HLA-A2/Tax₁₁₋₁₉complexes. Correctly folded HLA-A2/Tax₁₁₋₁₉ complex was isolated andpurified by anion exchange Q-Sepharose chromatography (Pharmacia)followed by site specific biotinylation using the BirA enzyme (Avidity,Denver, Colo.), as previously described (Altman J. D. et al., 1996.Science 274:94-96). The homogeneity and purity of the HLA-A2/Tax₁₁₋₁₉complex were analyzed by various biochemical means including SDS-PAGE,size exclusion chromatography, and enzyme linked immunosorbent assay(ELISA), as previously described (Denkberg, G. et al., 2000. Eur. J.Immunol. 30:3522-3532).

Selection of Fab-Phages Capable of Specifically Binding HLA-A2/Tax₁₁₋₁₉Complex:

Selection of Fab-phages (Fab-phages) on surface immobilized biotinylatedMHC/peptide complex was performed as previously described (Denkberg, G.et al., 2002. Proc. Natl. Acad. Sci. U.S.A. 99:9421-9426; Lev, A. etal., 2002. Cancer Res. 62:3184-3194). Briefly, a large human Fab librarycontaining 3.7×10¹⁰ different Fab clones (de Haard, H. J. et al., 1999.J Biol. Chem. 274:18218-18230) was used for the selection. Aliquots of10¹³ phages were pre incubated with 200 microliters of streptavidincoated paramagnetic beads (Dynal, Oslo) to deplete streptavidin binders.The remaining phages were then panned using decreasing amounts ofsurface immobilized biotinylated HLA-A2/Tax₁₁₋₁₉ complex as bindingtarget (500 nanomolar for the first round, and 100 nanomolar for thefollowing rounds). Bound phages were eluted with 100 millimolartriethylamine, and the eluate was neutralized with 1 molar Tris.HCl pH7.4. Neutralized phages were then used to infect E. coli TG1 cells(OD₆₀₀=0.5) by incubation for 30 minutes at 37 degrees centigrade.

The diversity of the selected antibodies was determined by DNAfingerprinting. The Fab DNA of different clones was PCR amplified usingthe primers pUC-reverse [5′-AGCGGATAACAATTTCACACAGG-3′ (SEQ ID NO: 1)]and fd-tet-seq24 [5′-TTTGTCGTCTTTCCAGACGTTAGT-3′ (SEQ ID NO: 2)]followed by digestion with BstNI (New England Biolabs, U.S.A.) byincubation for 2 hours at 60 degrees centigrade. Reaction products wereanalyzed by agarose gel electrophoresis.

Expression and Purification of Soluble Recombinant Fab's:

Fab's were expressed and purified as previously described (Denkberg, G.et al., 2000. Eur. J. Immunol. 30:3522-3532). Briefly, cultures of TG1or BL21 cells transformed with constructs for expression of Fab's underthe control of isopropyl beta-D-thiogalactoside (IPTG) inducibleregulatory sequences were grown to OD₆₀₀=0.8 to 1.0. Cultures wereinduced to express recombinant Fab by addition of 1 millimolar IPTG andfurther culturing for 3 to 4 hours at 30 degrees centigrade. Periplasmiccontent was released using B-PER solution (Pierce), and applied onto apre-washed TALON column (Clontech). Bound Fab was eluted from the columnusing 0.5 ml of 100 millimolar imidazole dissolved in phosphate bufferedsaline solution, and dialyzed twice against phosphate buffered salinesolution by overnight incubations at 4 degrees centigrade to removeresidual imidazole.

ELISA of Fab Phage Clones and Purified Fab's:

The binding specificities of individual Fab-phage clones and solubleFab's for HLA-A2/Tax₁₁₋₁₉ complex were determined by ELISA usingbiotinylated HLA-A2/Tax₁₁₋₁₉ complex as binding target. ELISA plates(Falcon) were coated overnight with BSA-biotin (1 microgram/well).Coated plates were washed and incubated for 1 hour at room temperaturewith streptavidin (1 microgram/well). After extensive washing, theplates were incubated for 1 hour at room temperature with 0.5 microgramof HLA-A2/Tax₁₁₋₁₉ complex. The plates were blocked for 30 minutes atroom temperature with 2 percent skim milk-phosphate buffered salinesolution, and were subsequently incubated for 1 hour at room temperaturewith about 10⁹ phage clones per well, or with various concentrations ofsoluble purified Fab. The plates were washed and incubated withhorseradish peroxidase conjugated anti human Fab antibody for solubleFab, or with horseradish peroxidase conjugated anti M13 phage antibodyfor Fab-phages. Detection was performed using TMB reagent (Sigma). Theamino acid sequences of the Tax₁₁₋₁₉ target peptide and of HLA-A2restricted negative control peptides used for screening the Fab-phageclones or purified Fab's are shown in Table 1.

TABLE 1 HLA-A2 restricted peptides used for screening Fab-phage clonesor purified soluble Fab's. Sequence* Protein Peptide position LLFGYPVYVTAX 11-19 (SEQ ID NO: 3) LLLTVLTVV MUC1-D6 13-21 (SEQ ID NO: 4)NLTISDVSV MUC1-A7 130-138 (SEQ ID NO: 5) NLVPMVATV CMV-pp65 495-503 (SEQID NO: 6) SVRDRLARL EBNA-3A 596-604 (SEQ ID NO: 7) ILAKFLHWL hTERT540-548 (SEQ ID NO: 8) RLVDDFLLV hTERT 865-873 (SEQ ID NO: 9) IMDQVPFSVGp100 209-217 (SEQ ID NO: 10) YLEPGPVTV Gp100 280-288 (SEQ ID NO: 11)KTWGQVWQV Gp100 154-162 (SEQ ID NO: 12) EAAGIGILTV MART 26-35 (SEQ IDNO: 13)

Production of Fluorescent Fab T3F2 Tetramer:

The genes encoding the light and heavy chains of Fab T3F2 were clonedseparately into a pET expression vector for T7-promoter regulatedexpression of cloned inserts. The light chain gene was engineered as afusion protein including the BirA recognition sequence for site specificbiotinylation at the carboxy terminus (T3F2 light-BirA). Theseconstructs were expressed separately in E. coli BL21 cells and uponinduction with IPTG, intracellular inclusion bodies containing largeamounts of the recombinant protein were generated. Inclusion bodiescontaining the T3F2 chains were purified, solubilized, reduced, andrefolded in-vivo at a 1:1 ratio in a redox shuffling buffer systemcontaining 0.1 molar Tris.HCl, 0.5 molar arginine, and 90 micromolaroxidized glutathione at pH 8.0. Correctly folded Fab was then isolatedand purified by anion exchange MonoQ chromatography (Pharmacia). The Fabpeak fractions were concentrated using Centricon-30 (Amicon) to 1milligram per milliliter and the buffer was exchanged to 10 millimolarTris.HCl pH 8.0. Biotinylation was performed using the BirA enzyme(Avidity, Denver, Colo.), as previously described (Denkberg, G. et al.,2000. Eur. J. Immunol. 30:3522-3532; Altman J. D. et al., 1996. Science274:94-96). Excess biotin was removed from biotinylated Fab using a G-25desalting column. Phycoerythrin labeled streptavidin(Jackson-Immunoresearch) was added at a molar ratio of 1:4 to producefluorescent tetramers of the biotinylated Fab.

Flow Cytometry:

The B cell line RMA-S-HHD transformant expressingHLA-A2-β₂-microglobulin, the EBV transformed HLA-A2 positive JY cells,mature human HLA-A2 positive dendritic cells, and the HLA-A2 negative Bcell line APD-70 were used to determine the reactivity of therecombinant Fab's with cell surface expressed HLA-A2/Tax₁₁₋₁₉ complex.Peptide pulsing was performed as indicated. Briefly, about 10⁶ cellswere washed twice with serum-free RPMI and incubated overnight at 26degrees centigrade or 37 degrees centigrade, respectively, in mediumcontaining 1 to 50 micromolar of the peptide. The RMA-S-HHD cells weresubsequently incubated at 37 degrees centigrade for 2 to 3 hours tostabilize cell surface expression of HLA-A2/Tax₁₁₋₁₉ complex.

Alternatively, 20×10⁶ JY or APD cells were transfected with 20micrograms of the eukaryotic expression vector pcDNA 3.1 (Invitrogen)encoding the TAX protein cDNA (pcTAX). The cDNA was a kind gift of Drs.M. Yutsudo (Osaka University) and T. Oka, (Okayama University). Twelveto twenty four hours after transfection, cells were incubated for 60 to90 minutes at 4 degrees centigrade with recombinant Fab (20 microgramper milliliter) in a volume of 100 microliters. After incubation, theprimarily labelled cells were washed three times, and incubated with 1microgram of anti human Fab antibody (Jackson-Immunoresearch). Thesecondarily labeled cells were then washed three times, and resuspendedin ice cold phosphate buffered saline solution. All subsequent washesand incubations were performed under ice cold conditions. The cells wereanalyzed using a FACStar flow cytometer (Becton Dickinson) and theresults were analyzed using WINANOMOLARDI software (Trotter J.,http://facs.scripps.edu). Flow cytometric analysis of peptide loadedcells was performed as previously described (Denkberg, G. et al., 2002.Proc. Natl. Acad. Sci. U.S.A. 99:9421-9426; Lev, A. et al., 2002. CancerRes. 62:3184-3194).

Competition Binding Assays:

Flexible ELISA plates were coated with BSA-biotin and 10 micrograms ofHLA-A2/Tax₁₁₋₁₉ complex in a volume of 100 microliters were immobilized,as previously described (Lev, A. et al., 2002. Cancer Res. 62:3184-3194;Cohen, C J. et al., 2002. Cancer Res. 62:5835-5844). Binding of solublepurified Fab was performed via a competitive binding analysis in whichthe ability of purified Fab to inhibit the binding of [125]iodine-Fab tospecific surface immobilized HLA-A2/Tax₁₁₋₁₉ complex was examined.Recombinant Fab was radiolabeled with [125]iodine using theBolton-Hunter reagent. The radiolabeled Fab was added to the wells as atracer (3×10⁵ to 5×10⁵ counts per minute per well) in the presence ofincreasing concentrations of unlabeled Fab as competitor. Binding assayswere performed by incubation at room temperature for 1 hour in phosphatebuffered saline solution. After incubation, plates were washed 5 timeswith phosphate buffered saline solution and bound radioactivity wasdetermined using a gamma counter. The apparent affinity of Fab wasdetermined by extrapolating the concentration of competitor necessary toachieve 50 percent inhibition of binding of [125]iodine labeled Fab tothe immobilized HLA-A2/Tax₁₁₋₁₉ complex. Non specific binding wasdetermined by adding a 20 to 40 fold excess of unlabeled Fab.

Enzyme-Linked Immunohistochemical Analysis of Specific Human MHC/ViralPeptide Complexes:

JY or APD cells were transfected with pcTAX vector, as described above.After 24 hours, transfected cells were incubated with 20 micrograms ofhorseradish peroxidase (HRP) labeled T3F2 Fab tetramer for 1 hour on icein RMPI supplemented with 10 percent FCS. The cell suspension wasapplied onto glass slides precoated with 0.1 percent poly-L-lysine(Sigma), as previously described [Harlow, E., and Lane, D. in:“Antibodies: A Laboratory Manual”. Cold Spring Harbor, Cold SpringHarbor Laboratory Press (1988)], and the slides were incubated for 1hour at room temperature. Following incubation, the slides were washedthree times with phosphate buffered saline solution, and incubated witha DAB+ solution (Dako) for 1 minute followed by washing with phosphatebuffered saline solution to remove excess staining reagent.

Expression and Purification of Soluble Recombinant Anti HLA-A2/Tax₁₁₋₁₉Complex Immunotoxin:

The DNA sequences encoding the heavy and light chain variable domains ofT3F2 were recovered from Fab-phage clone by PCR amplification andsubcloned using the NcoI-NotI fragment into bacterial expression vectorpIB-NN, for expression of T3F2-PE38, a single chain immunotoxinconsisting of the toxin PE38 KDEL fused to a single chain Fv of T3F2 viathe carboxy terminus of the light chain variable region. Toxin PE38 KDELconsists of the translocation and ADP-ribosylation domains ofPseudomonas exotoxin A. Expression in BL21 IDE3 cells, refolding frominclusion bodies, and purification of the T3F2-PE38 was performed aspreviously described (Brinkmann U. et al., 1991. Proc. Natl. Acad. Sci.U.S.A. 88:8616-20).

Experimental Results:

Generation of Anti HLA-A2/Tax₁₁₋₁₉ Complex Antibodies:

The immune response in HTLV-1 infected patients carrying the MHC class Iallele HLA-A2 is primarily directed against the HLA-A2 restricted Taxprotein derived Tax₁₁₋₁₉ peptide by clonal expansion of HTLV-1 reactiveCD8 positive T lymphocytes.

Recombinant HLA-A2/Tax₁₁₋₁₉ complex was generated using a previouslydescribed single chain MHC-β₂-microglobulin fusion protein expressionconstruct (Denkberg, G. et al., 2000. Eur. J. Immunol. 30:3522-3532).Using this construct, the extracellular domains of HLA-A2 are fused toβ₂-microglobulin using a flexible 15 amino acid long peptide linker. TheHLA-A2/Tax₁₁₋₁₉ complex was produced by in-vitro refolding of inclusionbodies in the presence of Tax₁₁₋₁₉ peptide. The refolded HLA-A2/Tax₁₁₋₁₉complex was found to be very pure, homogenous, and monomeric, asdetermined by SDS-PAGE and size-exclusion chromatography analyses (datanot shown). Recombinant HLA-A2/Tax₁₁₋₁₉ complex generated by thisstrategy has been previously characterized in detail with respect to itsbiochemical, biophysical, and biological properties, and was found to becorrectly folded and functional [Denkberg, G. et al., 2000. Eur. J.Immunol. 30:3522-3532; Harlow, E., and Lane, D. in: “Antibodies: ALaboratory Manual”. Cold Spring Harbor: Cold Spring Harbor LaboratoryPress (1988)].

For selection of antibodies capable of specifically binding a specificMHC/peptide complex, a large Fab-phage library consisting of arepertoire of 3.7×10¹⁰ recombinant human Fab's (de Haard, H. J. et al.,1999. J. Biol. Chem. 274:18218-18230) was used. Due to exposure of theFab's to streptavidin coated plates during selection, the library wasfirst depleted of streptavidin binders, and subsequently used forpanning soluble recombinant HLA-A2/Tax₁₁₋₁₉ complex. A 1,300 foldenrichment in phage titer was observed after three rounds of panning(Table 2). The specificity of the selected Fab-phages was determined bya differential ELISA using streptavidin coated wells incubated withbiotinylated HLA-A2 in complex with either the Tax₁₁₋₁₉ peptide ornegative control HLA-A2 restricted peptides. Phage clones analyzedfollowing the third round of selection exhibited two types of bindingpatterns toward the HLA-A2/Tax₁₁₋₁₉ complex; one class of antibodiesconsisted of pan MHC binders which cannot differentiate between thevarious specific MHC/peptide complexes; the second type consisted ofantibodies that specifically bound the HLA-A2/Tax₁₁₋₁₉ complex. TheELISA screen revealed that 87 percent of randomly selected clones(78/90) screened from the third round of panning appeared tospecifically bind the HLA-A2/Tax₁₁₋₁₉ complex.

TABLE 2 Screening of Fab-phages for anti HLA-A2/Tax₁₁₋₁₉ complex Fab's.Fraction Phage Phage Ratio Fold MHC/peptide MHC/peptide No. of Cycleinput (I) output (O) (O/I) enrichment complex binders complex bindersFab's 1 7.2 × 10¹² 9.6 × 10⁵ 1.3 × 10⁻⁷ — — — — 2 5.8 × 10¹³ 1.1 × 10⁷1.9 × 10⁻⁷ 1.5 15/90 (17%) 10/90 (11%) 6 3 5.2 × 10¹³ 8.7 × 10⁹ 1.7 ×10⁻⁴ 1,300 78/90 (87%) 56/90 (62%) 14

However, an unexpectedly high percentage of Fab's, 62 percent (56/90),were fully Tax₁₁₋₁₉ peptide dependent for binding and specific forHLA-A2/Tax₁₁₋₁₉ complex when tested as Fab-phages in ELISAs usingvarious HLA-A2/control peptide complexes as binding targets. As shown inTable 2, 62 percent of the clones bound only to the HLA-A2/Tax₁₁₋₁₉complex and not to negative control complexes containing other HLA-A2restricted peptides. Such clones thus exhibited an MHC restrictedpeptide specific binding similar to T-cell receptors (TCRs). Theseapparent HLA-A2/Tax₁₁₋₁₉ complex specific clones remained specific forHLA-A2/Tax₁₁₋₁₉ complex in a secondary screening using HLA-A2 complexedwith other HLA-A2 restricted peptides (listed under Materials andMethods). FIG. 1 shows a representative analysis of four Fab cloneswhich reacted only with the HLA-A2/Tax₁₁₋₁₉ complex and not withHLA-A2/negative control peptide complexes displaying melanoma gp100 andMART-1 derived epitopes, and the MUC1 derived D6 epitope.

The diversity pattern of the peptide specific clones (from round two orthree) was examined by DNA fingerprint analysis. Twenty differentrestriction patterns (6 for clones isolated from the second round ofpanning, and 14 different patterns after the third round of selection)were found, indicating successful selection of several different Fab'scapable of specifically binding HLA-A2/Tax₁₁₋₁₉ complex. DNA sequencinganalysis confirmed these observations. The variable heavy and variablelight chain complementarity determining region sequences of 14 clonesspecific for HLA-A2/Tax₁₁₋₁₉ complex are shown in Table 3.

TABLE 3 Amino acid sequences of complementarity determining regions ofFab's specifically binding HLA-A2/Tax₁₁₋₁₉ complex. Fab Chain* CDR1 CDR2CDR3 T3E3 H SYTIS GIIPIFGTANYAQKFQG DTDSSGYYGAVDY (SEQ ID NO: 14) (SEQID NO: 15) (SEQ ID NO: 16) L RASQSVGSYLA DASHRAT QQRSNWPPMYT (SEQ ID NO:17) (SEQ ID NO: 18) (SEQ ID NO: 19) T3F2 H SYGMH VISYDGSNKYYADSVKGDFDYGDSYYYYGMDV (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) LRSSQSLLHSNGY LGSNRAS MQALQTPRT (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ IDNO: 25) T3D4 H NYGIN WISAYNGDTKYAQRLQD GDSTVGYEYLQY (SEQ ID NO: 26) (SEQID NO: 27) (SEQ ID NO: 28) L QASQGIGKYLN VASSLQS QQTSSFPPT (SEQ ID NO:29) (SEQ ID NO: 30) (SEQ ID NO: 31) T3D3 H SYAIS RIIPILGIANYAQKFQGQGGDYSNYYYYMDV (SEQ ID NO: 32) (SEQ ID NO: 33) (SEQ ID NO: 34) LRASQSVSSYLA DASNRAT QHRFNWPVT (SEQ ID NO: 35) (SEQ ID NO: 36) (SEQ IDNO: 37) T3D1 H SYGMH VISYDGSNKYYADSVKG DQTYYGSGSPRGGLDY (SEQ ID NO: 38)(SEQ ID NO: 39) (SEQ ID NO: 40) L TGSSGSIANNYVQ EDDQRPS QSYDNSNSFVV (SEQID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) T2B12 H SNSAAWNRTYYRSKWYNDYVSVKS GPYDTTGPWGNWFDP (SEQ ID NO: 44) (SEQ ID NO: 45) (SEQID NO: 46) L RASQSVSSDLA GASYRAT QQYGSSPRT (SEQ ID NO: 47) (SEQ ID NO:48) (SEQ ID NO: 49) T2G7 H SYGMH VISYDGSNKYYADSVKG DFDYGDSYYYYGMDV (SEQID NO: 50) (SEQ ID NO: 51) (SEQ ID NO: 52) L RSSQSLLHSNGYNYLD LGSNRASMQALQTPRT (SEQ ID NO: 53) (SEQ ID NO: 54) (SEQ ID NO: 55) T2H9 H SYAMSAISGSGGSTYYADSVKG DSLAGATGTDFDY (SEQ ID NO: 56) (SEQ ID NO: 57) (SEQ IDNO: 58) L RASQTVTANYLA DASVRAT QQYGSSPIT (SEQ ID NO: 59) (SEQ ID NO: 60)(SEQ ID NO: 61) T3A2 H SYAMS GISGSGGSTYYADSVKG DFDYGGNSGSLFDY (SEQ IDNO: 62) (SEQ ID NO: 63) (SEQ ID NO: 64) L GASESVGGNYLA DASTRATQHYGSSPSTY (SEQ ID NO: 65) (SEQ ID NO: 66) (SEQ ID NO: 67) T3A4 H SSNWWSEIYHSGSTNYNPSLKS HSYDYLWGTYRFDY (SEQ ID NO: 68) (SEQ ID NO: 69) (SEQ IDNO: 70) L RASQDIGTWLA AATTLES QQARSLPYT (SEQ ID NO: 71) (SEQ ID NO: 72)(SEQ ID NO: 73) T3B5 H NYGIN WISAYNGDTKYAQRLQD GDSTVGYEYLQY (SEQ ID NO:74) (SEQ ID NO: 75) (SEQ ID NO: 76) L QASQGIGKYLN VASSLQS QQTSSFPPT (SEQID NO: 77) (SEQ ID NO: 78) (SEQ ID NO: 79) T4B7 H SYGMHVISYDGSNKYYADSVKG DYNGYGDYVLGY (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ IDNO: 82) L RASQSVSSYLA DASNRAT QQRSNWASYT (SEQ ID NO: 83) (SEQ ID NO: 84)(SEQ ID NO: 85) T4D10 H SYYMH IINPSGGSTSYAQKFQG DRGGGYDVSPYGMDV (SEQ IDNO: 86) (SEQ ID NO: 87) (SEQ ID NO: 88) L RASQSISSYLN AASNLQT QQTYSLPT(SEQ ID NO: 89) (SEQ ID NO: 90) (SEQ ID NO: 91) T4B12 H SYAISGIIPIPGITNYAQKFQG RVGYYYGMDV (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO:94) L AGSGGDIASNFVQ EENRRPS QSYDGSAW (SEQ ID NO: 95) (SEQ ID NO: 96)(SEQ ID NO: 97) *H - heavy chain, L - light chain

Specificity and Affinity of Anti HLA-A2/Tax₁₁₋₁₉ Complex Fab's:

Using E. coli BL21 or TG1 cells, soluble Fab's from 3 phage clonesexhibiting the most specific binding pattern to HLA-A2/Tax₁₁₋₁₉ complex(analyzed above, FIG. 1) were produced.

SDS-PAGE analysis of Fab's purified from the periplasmic fraction of E.coli transformants by nickel affinity chromatography revealedhomogenous, pure Fab's with the expected molecular weight of 50 kDa(FIG. 2 a). Quantities of 2 to 4 milligrams of pure Fab was obtainedfrom 1 liter of bacterial shake flask culture. For further manipulation;i.e. to increase the avidity of monomeric Fab's, the Fab's were alsoproduced by in-vitro refolding. The light chain and Fd fragment(truncated portion of the heavy chain consisting of the variable regionand the CH1 domain of the constant region) were subcloned into pET basedexpression vectors for T7 promoter regulated expression of clonedinserts, and upon induction with IPTG, large amounts of recombinantprotein accumulated as intracellular inclusion bodies (FIG. 2 b). Uponin-vitro redox shuffling refolding, purified monomeric Fab's wereobtained in high yield (4 to 6 milligrams of purified Fab was obtainedfrom two 1 liter shake flask cultures, each expressing the Fab light orFd fragment; FIG. 2 c).

The fine specificity of the soluble Fab's for HLA-A2/Tax₁₁₋₁₉ complexwas analyzed by ELISA using biotinylated HLA-A2/Tax₁₁₋₁₉ compleximmobilized to BSA-streptavidin coated wells. TheBSA-streptavidin-biotin spacer enables the correct folding of thecomplex, which may be distorted by direct binding to plastic. To verifycorrect folding of the bound complex and its stability during bindingassays, the ability of the bound complex to react with the conformationspecific monoclonal antibody w6/32 which exclusively recognizescorrectly folded, peptide complexed HLA was monitored. FIGS. 3 a-c showspecific binding of soluble Fab's T3D4, T3E3, and T3F2, respectively, toHLA-A2/Tax₁₁₋₁₉ complex, but not to 10 control HLA-A2/peptide complexescontaining viral epitopes derived from CMV or EBV, and a variety oftumor associated epitopes such as telomerase epitopes (540, 865),melanoma gp100 and MART-1 derived epitopes (154,209,280 and MART,respectively), and the MUC1 derived epitopes A7 and D6 (see experimentalprocedures for list of peptides). Thus, these anti specific MHC/peptidecomplex Fab's exhibit the binding characteristics and fine specificityof a TCR. In control experiments, the Fab's did not recognize theTax₁₁₋₁₉ peptide alone when immobilized on the plate, nor immobilizedstreptavidin or other protein antigens such as BSA, IgG, RNAse, orchymotrypsin (data not shown).

The binding affinity properties of two of the soluble Fab's were testedusing a saturation ELISA assay using addition of increasing amounts ofFab's to biotinylated HLA-A2/Tax₁₁₋₁₉ bound to streptavidin coatedplates. As shown in FIGS. 4 a-b, the binding of Fab's T3E3 and T3F2,respectively, was dose dependent and saturable. Extrapolating the 50percent binding signal of either fragment revealed that their affinitywas in the nanomolar range.

Finally, the apparent binding affinity of the Fab's for HLA-A2/Tax₁₁₋₁₉complex was determined using a competition binding assay in which thebinding of [125]iodine labeled Fab was competed with increasingconcentrations of unlabeled Fab's. These binding studies (FIG. 4 c)revealed an apparent binding affinity of approximately 25 to 30nanomolar for the T3F2 antibody. Similar results were observed for theT3E3 antibody (not shown).

Detection of HLA-A2/Tax₁₁₋₁₉ Complex on Peptide Pulsed AntigenPresenting Cells (APCs):

To demonstrate that the isolated Fab's can specifically bindHLA-A2/Tax₁₁₋₁₉ complex not only in the recombinant soluble form butalso in the native form, as expressed on the cell surface, murine TAP2deficient RMA-S cells transfected with the human HLA-A2 gene in a singlechain format (Pascolo, S. et al., 1997. J. Exp. Med. 185:2043-2051)(HLA-A2.1/Db-β₂-microglobulin single chain, RMA-S-HHD cells). TheTax₁₁₋₁₉ peptide and HLA-A2 restricted control peptides were loaded onRMA-S-HHD cells and the ability of the selected Fab's to bind to peptideloaded cells was monitored by flow cytometry. Peptide induced MHCstabilization of the TAP2 mutant RMA-S-HHD cells was demonstrated byreactivity of monoclonal antibodies w6/32 (HLA conformation dependent)and BB7.2 (HLA-A2 specific) with peptide loaded but not unloaded cells(data not shown). Fab's T3E3 and T3F2 reacted only with Tax₁₁₋₁₉ peptideloaded RMA-S-HHD cells but not with cells loaded with the gp100 derivedG9-154 peptide (FIGS. 5 a-b, respectively). Similar results wereobserved using flow cytometric analysis using 10 other HLA-A2 restrictedcontrol peptides (data not shown).

Cells of the TAP and HLA-A2 positive EBV transformed B lymphoblast cellline JY were also used as APCs. The cells were incubated with Tax₁₁₋₁₉peptide, and HLA-A2 restricted control peptides, and followingincubation the cells were washed and incubated with the Fab's. The T3E3or T3F2 Fab's were found to bind only to JY cells incubated with theTax₁₁₋₁₉ peptide against which they were selected but not to HLA-A2restricted control peptides (FIGS. 5 c-d, respectively). As a control,peptide loaded HLA-A2 negative/HLA-A1 positive APD B cells were alsoused. No binding of the Fab's to these cells was observed (data notshown). Fab's T3E3 and T3F2 were also tested for binding to peptidepulsed mature HLA-A2 positive dendritic cells. As shown in FIGS. 5 e-f,respectively, the T3E3 and T3F2 Fab's recognized HLA-A2 positivedendritic cells pulsed with Tax₁₁₋₁₉ peptide but not with a controlgp100 derived peptide.

The Fab's were modified for detection of MHC/peptide complex on thesurface of cells. Since the density of a particular endogenousHLA/peptide complex on cells is expected to be low compared to that ofpeptide pulsed APCs, the avidity of Fab T3F2 was increased by making Fabtetramers, which are directly tagged with a fluorescent probe. Thisapproach was used previously to increase the binding avidity ofMHC/peptide complexes to TCRs or to increase the sensitivity ofrecombinant antibody molecules (Cloutier, S. M. et al., 2000. Mol.Immunol. 37:1067-1077). Another advantage of using fluorescently labeledtetramers is that only a single staining step is required, whereasmonomeric unlabeled Fab's require a fluorescently labeled secondaryantibody. The Fab tetramers generated with fluorescently labelledstreptavidin were thus used to measure the expression of HLA-A2/Tax₁₁₋₁₉complex on the surface of peptide pulsed APCs. As shown in FIGS. 6 a-c,the intensity of the binding as measured by flow cytometry with peptidepulsed RMA-S-HHD (FIG. 6 a), JY cells (FIG. 6 b), and human dendriticcells (FIG. 6 c), was dramatically increased by two logs compared to thestaining intensity with the T3F2 Fab monomer.

Unexpectedly, the staining pattern of the mature HLA-A2 positivedendritic cells was found to be scattered over a wide range offluorescence intensities, indicating for the first time that dendriticcell populations display heterogeneous levels of specific MHC/peptidecomplexes at the cell surface. Such results therefore indicate thepotency of the Fab's such as those described herein for studying thebiology of specific MHC/peptide complex presentation by APCs.

In particular, these results demonstrate the ability of the Fab's todetect cell surface displayed HLA-A2/Tax₁₁₋₁₉ complex.

Cell Surface Detection of HLA-A2/Tax₁₁₋₁₉ Complex Formed byIntracellular Antigen Processing:

To examine the ability of the Fab's to detect HLA-A2/Tax₁₁₋₁₉ complexproduced by physiological antigen processing, the HTLV-1 Tax gene wastransfected into HLA-A2 positive and negative JY or APD cells,respectively. Twenty four hours following transfection, the reactivityof T3F2 to cell surface displayed HLA-A2/Tax₁₁₋₁₉ complex was tested byflow cytometry. The analysis was performed using the high aviditytetrameric Fab T3F2. Positive staining above control could be clearlyseen only with HLA-A2 positive JY cells transfected with the Tax genebut not with HLA-A2 negative cells transfected with the Tax gene (FIGS.7 a-b, respectively). Negative control Fab G2D12 specific forHLA-A2/G9-154 complex did not react with the Tax transfected JY cells(FIG. 7 a). The Tax₁₁₋₁₉ peptide specific, MHC restricted pattern ofreactivity by T3F2 was not due to differences in transfectionefficiency, or HLA expression of JY and APD cells. As determined viacontrol experiments employing transfection of green fluorescent protein(GFP) into these cells, the percentage of transfected cells with bothcell lines using the same transfection protocol used for expression ofFab was similar (FIG. 7 c), and the staining intensity of these cellswith w6/32, a pan MHC monoclonal antibody, was similar (data not shown).These results indicate that the Fab's are capable of detectingHLA-A2/Tax₁₁₋₁₉ complex formed by intracellular antigen processing.

The use of Fab T3F2 for detecting HLA-A2/Tax₁₁₋₁₉ complex on virusinfected cells was attempted. To this end, HLA-A2 negative HUT 102 andHLA-A2 positive RSCD4 cells (human CD4 positive T lymphocyte cell linesinfected with HTLV-1) were used. As shown in FIG. 7 d, a significantstaining with Fab T3F2 was observed on RSCD4 but not on HUT 102 cells,indicating that the Fab is capable of detecting the specificHLA-A2/Tax₁₁₋₁₉ complex on the surface of virus infected cells.Unexpectedly, the staining pattern revealed two cell subpopulationshaving moderate or high reactivity, respectively, with the Fab, whichmay indicate variability in the expression of the HLA-A2/Tax₁₁₋₁₉complex within subpopulations of RSCD4 HTLV-1 infected cells. Similarvariability was observed in staining experiments with an anti Taxprotein antibody (not shown). Negative control Fab G2D12 specific forHLA-A2/G9-154 complex did not stain RSCD4 cells (FIG. 7 d).

These results underscore the utility of anti specific MHC/peptidecomplex Fab's, in particular that of the above described antiHLA-A2/Tax₁₁₋₁₉ complex fragments, for the study of antigen presentationon APCs as well as virus infected cells.

High Sensitivity Detection and Direct Quantitation of Surface ExpressedHLA-A2/Tax₁₁₋₁₉ Complex on APCs and Virus Infected Cells:

The data presented above demonstrate the high specificity of theHLA-A2/Tax₁₁₋₁₉ complex specific Fab's as well as their ability todetect naturally processed Tax₁₁₋₁₉ peptide complexed with HLA-A2. Thesensitivity of specific MHC/peptide recognition by the Fab's in-vitrowas tested by staining with Fab T3F2 was tested over a broad range ofTax₁₁₋₁₉ peptide concentrations. As shown in FIGS. 8 a-b, titration ofpeptide pulsed JY cells using graded concentrations of Tax₁₁₋₁₉ peptidedemonstrated staining intensity dependent on the concentration of thepeptide used for pulsing, and that the Fab was capable of detectingHLA-A2-Tax₁₁₋₁₉ complex when pulsing Tax₁₁₋₁₉ peptide at a concentrationin the low nanomolar range. The staining intensity of peptide pulsed JYcells observed with T3F2 Fab was estimated by comparison to calibrationbeads displaying graded numbers of phycoerythrin molecules. Thiscomparison enabled determination of the number of HLA-A2/Tax₁₁₋₁₉complexes displayed on the surface of cells that are pulsed with variousconcentrations of the Tax₁₁₋₁₉ peptide (FIG. 8 a and Table 2). Specificdetection of as few as 100 HLA-A2-Tax₁₁₋₁₉ complexes per cell wasachieved (using 6 nanomolar Tax₁₁₋₁₉ peptide pulsing) and reachedsaturation at about 1.1× to 1.2×10⁵ complexes per cell when pulsing with25 to 50 micromolar Tax₁₁₋₁₉ peptide.

These results therefore demonstrate that the sensitivity of specificMHC/peptide complex detection by T3F2 Fab is in the same range as theminimal concentration peptide needed to elicit measurable cytokinesecretion (IL-2 or IFN-γ) from T lymphocyte hybridomas or target Tlymphocyte lysis by CD8 positive cytotoxic T lymphocyte lines (Reis eSousa, C., and Germain, R. N., 1995. J. Exp. Med. 182:841-851; Reis eSousa, C. et al., 1996. J. Exp. Med. 184:149-157).

A major problem hampering the study of MHC dependent antigenpresentation is the unavailability of adequate methods for quantifyingsurface expression levels on individual cells of specific MHC/peptidecomplexes produced by intracellular antigen processing. Using flowcytometric analysis of cell surface display of HLA-A2/Tax₁₁₋₁₄ complexusing Fab T3F2 and comparison of the fluorescence intensity of T3F2stained cells with that of calibration beads displaying graded numbersof phycoerythrin sites, it was possible to quantitate the number ofspecific HLA-A2/Tax₁₁₋₁₉ complexes on the cell surface (Table 4).Namely, JY cells pulsed with 1.5 micromolar Tax₁₁₋₁₉ peptide displayedon their surface 5×10³ complexes per cell, while JY cells transfectedwith the Tax gene displayed on their surface, after intracellularantigen processing, 1×10⁴ complexes per cell. The latter result is incomplete agreement with recent quantitation of murine H-2k^(b) bound tothe ovalbumin peptide SIINFEKL after recombinant Vaccinia virusinfection of cells in-vitro using an anti specific mouse MHC/peptidecomplex antibody (Porgador, A. et al., 1997. Immunity 6:715-726). Asshown in FIG. 7 d and Table 4, direct detection of HLA-A2/Tax₁₁₋₁₉complex on HTLV-1 infected cells enabled quantification of the number ofcomplexes displayed on these cells. This analysis, using calibrationbeads, revealed that virus infected RSCD4 cells display on their surfaceabout 3×10⁴ HLA-A2/Tax₁₁₋₁₉ complexes per cell. As demonstrated in FIG.7 d, Fab T3F2 recognized two subpopulations of HTLV-1 infected RSCD4cells with high and moderate reactivity. The highly reactive cellsexpress on their surface 3×10⁴ HLA-A2/Tax₁₁₋₁₉ complexes while the cellpopulation with low to moderate staining intensity expresses severalhundred HLA-A2/Tax₁₁₋₁₉ complexes. These results clearly demonstrate thepower of such anti specific MHC/peptide complex Fab's to quantitatespecific MHC/peptide complex expression on each cell in a population.

Detection of Cells Displaying HLA-A2/Tax₁₁₋₁₉ Complex in a HeterogeneousCell Population:

At present, there are no reagents available for detecting andphenotyping individual cells displaying specific MHC/peptide complexesin mixed cell populations. Such reagents would have great utility, forexample, for detecting or staging tumorigenic cells, or for studyingantigen presentation in lymphoid tissues within heterogeneous cellpopulations. The anti specific MHC/peptide complex Fab's described abovewould be ideally suited to conduct such analyses. To simulate aheterogeneous population of cells in which only a small fractionexpresses a specific MHC/peptide complex, Tax transfected and controlnon transfected JY cells were mixed in various ratios, and thereactivity of T3F2 Fab to such cells was analyzed by flow cytometry. Asshown in FIG. 8 c, single color flow cytometric analysis using T3F2 Faballows accurate identification of the admixed Tax transfected JY cellsthat express on their surface HLA-A2/Tax₁₁₋₁₉ complex generated byintracellular antigen processing. T3F2 Fab was shown to be able todetect Tax transfected JY cells in a proportion as low as 1 percentwithin a population of non transfected cells (FIGS. 8 c-d), asdemonstrated by the ability to detect 0.5 percent of positive cells(calculated from a maximal 61.2 percent transfection efficiency of JYcells; FIG. 8 d).

TABLE 4 Quantitation of the number of HLA-A2/Tax₁₁₋₁₉ complexes on thesurface of APCs and HTLV-1-infected cells Cells Mean number of sites percell* JY (50 mM peptide pulsed) 120,132 ± 16,934 JY (1.5 mM peptidepulsed) 5,150 ± 691  JY (Tax-transfected) 12,746 ± 2,877 RSCD4 (CD4positive T-cells, High: 32,820 ± 4,910 HTLV-1-infected) Low: 456 ± 72Background**  32 ± 13 *The fluorescence intensity of stained cells ineach experiment was compared with fluorescence intensities ofcalibration beads with known numbers of phycoerythrin (PE) molecules perbead (QuantiBRITE PE beads, Becton-Dickinson) and the number of sitesfor each experiment was determined. The mean number of non specificsites was determined by the intensity of staining of cells that areHLA-A2 positive but not infected with HTLV-1, HLA-A2 negative cellsinfected with HTLV-1, or APCs not transfected with the Tax gene. Thenumber of specific sites for each experiment was then calculated foreach experiment. The deviation in number of sites depend on thesensitivity of detection and the physiological status of the cells ineach individual determination. **The background number of sites wasdetermined as described, using SK-BR3 (HLA-A2 negative/HUT102), FM3D(HLA-A2 positive), and JY (HLA-A2 positive) cells not transfected withthe Tax gene as controls.

These results demonstrate the ease with which anti specific MHC/peptidecomplex Fab's can reveal a cell subpopulation bearing a specificendogenously generated MHC/peptide complex.

Immunohistochemical Detection of Cells Displaying HLA-A2/Tax₁₁₋₁₉Complex Generated by Intracellular Antigen Processing:

Another major potential use for anti specific MHC/peptide complexantibodies is in situ immunohistochemical analysis of specificMHC/peptide complexes in tissues. As a first step to assess thispotential, the capacity of T3F2 Fab to detect in situ HLA-A2/Tax₁₁₋₁₉complex displayed on JY cells by intracellular antigen processing wasdetermined. Tax transfected JY cells were subjected to single stepimmunohistochemical analysis using horseradish peroxidase conjugatedT3F2 Fab. As shown in FIGS. 9 a-f, these experiments showed the capacityof the Fab to strongly and specifically stain Tax transfected (FIGS. 9a-b) but not control non transfected JY cells (FIG. 9 c). Negativecontrol Fab G2D12 specific for HLA-A2/G9-154 complex did not exhibit anysignificant immune reactivity on Tax transfected JY cells (FIG. 9 d).Further evidence for the specific, MHC restricted reactivity of Fab T3F2in these in situ immunohistochemistry experiments is provided by thelack of reactivity of the Fab with Tax transfected (FIG. 9 e) and nontransfected (FIG. 9 f) HLA-A2 negative/HLA-A1 positive APD cells. Thesedata demonstrate the capacity of the T3F2 Fab to specifically detectHLA-A2/Tax₁₁₋₁₉ complex generated by intracellular antigen processing insitu on cells and potentially in tissue sections. To the presentinventors' knowledge, this is the first demonstration of in situdetection of a specific human MHC/peptide complex.

Specific Cytolysis of Cells Displaying HLA-A2/Tax₁₁₋₁₉ Complex byT3F2-PE38 Immunotoxin:

The capacity of an anti specific human MHC/viral peptide compleximmunotoxin to cytolyse cells displaying such a complex was determinedby testing the capacity of T3F2-PE38 to kill/damage peptide loaded APCs.The killing assay was performed by loading JY cells with Tax₁₁₋₁₉peptide, or control HLA-A2 restricted peptides, including the gp 100derived G9-209 peptide. As shown in FIG. 10, T3F2-PE38 was capable ofkilling JY cells loaded with Tax₁₁₋₁₉ peptide with an IC₅₀ of 2,500nanograms per milliliter. No T3F2-PE38 mediated cytolysis of JY cellsloaded with control HLA-A2 restricted peptides, or of cells not loadedwith peptide occurred.

Thus, the capacity to specifically and efficiently kill/damage targetcells displaying a specific human MHC/viral peptide complex usingcytotoxic conjugates targeted using an antibody specific for such acomplex was demonstrated for the first time.

Discussion:

The above described results demonstrate for the first time generation ofrecombinant antibody derived molecules, such as Fab's, capable ofspecifically binding specific human MHC/pathogen-derived peptidecomplexes, such as MHC/viral peptide complexes, and of cytotoxicconjugates including such molecules to specifically kill/damage cellsdisplaying such complexes. Until now, anti specific MHC/pathogen-derivedpeptide complex antibodies have been generated against murine forms ofsuch complexes only (Andersen, P. S. et al., 1996. Proc. Natl. Acad.Sci. U.S.A 93:1820-1824; Day, P. M. et al., 1997. Proc. Natl. Acad. Sci.U.S.A. 94:8064-8069; Porgador, A. et al., 1997. Immunity 6:715-726;Reiter, Y. et al., Proc. Natl. Acad. Sci. U.S.A. 94:4631-4636).

These novel molecules exhibit high affinity, high specificity binding tospecific human MHC/pathogen-derived peptide complexes, and hence displayTCR like specificity for such complexes. However, in contrast to theinherently low affinity of TCRs for MHC/peptide complexes, thesemolecules display the high affinity antigen binding characteristics ofantibodies, while retaining TCR specificity. By virtue of suchcharacteristics, such molecules have very promising utility in thenumerous diagnostic, therapeutic and scientific applications which wouldbenefit from the capacity to specifically label or target specific humanMHC/pathogen-derived peptide complexes such as those comprising viralpeptides.

Crucial features of these Fab's were identified, including the capacityto: (a) bind with high sensitivity and specificity particular humanMHC/pathogen-derived peptide complexes, such as HLA-A2/Tax₁₁₋₁₉ complex,expressed or displayed by cells which are infected with a pathogen suchas HTLV-1, peptide loaded, in suspension, and/or surface immobilizedusing immunohistochemical techniques; and (b) the capacity to delivermolecules, such as toxins, to cells displaying a specific humanMHC/pathogen-derived peptide complex, such as HLA-A2/Tax₁₁₋₁₉ complex.

An important feature of these molecules is their capacity to detectspecific human MHC/pathogen-derived peptide complexes at surfacedensities near the threshold limit required for triggering signaling viathe TCR. Studies from other laboratories using a monoclonal antibodyspecific for mouse MHC class I (H-2K^(b)) in complex with an ovalbuminpeptide indicated that the lower limit of sensitivity of flow cytometrydetection is in the range of 100 to 500 specific MHC/peptide complexesper cell using single step or sandwich staining techniques (Porgador, A.et al., 1997. Immunity 6:715-726). The data presented herein for antispecific human MHC/pathogen-derived peptide Fab's are in good agreementwith these numbers since the HLA-A2/Tax₁₁₋₁₉ complex specific Fab wasable to detect in a reproducible manner as few as 100 complexes percell. These numbers agree with several estimates of the threshold numberof specific MHC/peptide complexes on APCs required to elicit effectorresponses from T lymphocytes, such as cytokine secretion (Demotz, S. etal., 1990. Science 249:1028-1030; Harding, C. V., and Unanue, E. R.,1990. Nature 346:574-576), and are about 10 fold greater than what maybe required for cytotoxic T lymphocyte mediated cell lysis (Christinck ER. et al., 1991. Nature 352:67-70; Sykulev Y. et al., 1996. Immunity4:565-71). Using flow cytometry, it was possible to use an anti specifichuman MHC/pathogen-derived peptide complex Fab to detect such complexeson cells pulsed with peptide concentrations being similar to thoserequired to trigger cytokine secretion by T lymphocyte hybridoma orcytotoxic T lymphocyte lines, and being within a few fold ofconcentrations required for sensitizing target T lymphocytes for lysisin a short term assay by APCs (Porgador, A. et al., 1997. Immunity6:715-726). The presently described data indicate that when applied todissociated cell populations using flow cytometry, the Fab's can detectspecific human MHC/pathogen-derived peptide complexes at densitiesapproaching those required for activating T lymphocytes. Hence thesemolecules are suitable reagents for evaluating specific humanMHC/pathogen-derived peptide complex expression at low butphysiologically relevant levels.

This principle was applied here to mixtures of the parental JY APCs andits Tax gene transfected derivative. The latter intracellularlyprocesses Tax antigens and displays the relevant HLA-A2/Tax₁₁₋₁₉ complexat the cell surface, as demonstrated by positive staining of Taxtransfected but not control cells using T3F2 Fab. Even when using T3F2Fab in a single step staining for flow cytometry, it was possible toreadily identify Tax transfected cells admixed with non transfected JYcells in a proportion as low as 1 percent. The extreme ease with whichprecise quantitation of cell surface expressed specific humanMHC/pathogen-derived peptide complexes can be accomplished using thisapproach also makes it an invaluable tool for analyzing antigenprocessing and presentation. Increasingly, such analyzes are aimed atdetermining quantitative differences in antigen display resulting fromuse of distinct forms of an antigen, of various antigen deliverymethods, or of cells deficient in some known or suspected component ofthe antigen processing machinery. Without reagents such as the presentlydescribed anti specific human MHC/viral peptide complex Fab's, thequantitation of cell surface levels of specific humanMHC/pathogen-derived peptide complexes relies on biochemical isolationof antigenic peptides. This is an expensive and laborious processsubject to numerous experimental artifacts and cannot distinguishbetween intracellular pools of loaded molecules and those on the cellsurface accessible to TCRs.

In the data presented here, anti HLA-A2/Tax₁₁₋₁₉ complex Fab's enabledquantitation of such complexes generated by intracellular antigenprocessing on the surface of cells transfected with the Tax gene or onHTLV-1 infected cells. This analysis demonstrated that intracellularantigen processing in Tax transfected cells led to a display of about10⁴ specific MHC/peptide complexes per cell. Comparison with totalHLA-A2 staining showed that nearly 90 percent of the HLA-A2 moleculeswere occupied with a single peptide species (not shown). These dataagree with previous studies in which the number of H-2K^(b)/ovalbuminderived peptide complexes on the surface of cells following infectionwith recombinant Vaccinia virus encoding the peptide was analyzed in avariety of contexts (Porgador, A. et al., 1997. Immunity 6:715-726).These data also agree with results from studies investigating theoccupancy of MHC class I molecules by peptides derived from virallyencoded proteins displayed by infected cells (Anton, L. C. et al., 1997.J. Immunol. 158:2535-42). Such occupancy estimates were obtained byanalysis of stabilization of newly synthesized MHC class I heavychain-β₂-microglobulin complexes, or by elution of peptides fromexpressed MHC class I molecules and reconstruction experiments todetermine the peptide concentration in the eluates. The ability of FabT3F2 to detect the heterogeneity of HLA-A2/Tax₁₁₋₁₉ complex expressionlevels in a population of virally infected cells was shown. Such noveland striking data highlight the potential utility of such antibodies forstudying specific human MHC/pathogen-derived peptide complex expressionin contexts such as diagnosis of infection with a pathogen.

Immunohistochemical staining with T3F2 Fab permitted in situ detectionof HLA-A2/Tax₁₁₋₁₉ complex generated by intracellular antigen processingon the surface of Tax transfected JY cells. Staining of background HLAdisplay levels with the Fab was insignificant under these conditionsbecause neither non transfected cells nor HLA-A2 negative cellstransfected with Tax exhibited positive staining. Such data representthe first immunohistochemical visualization of a specific humanMHC/peptide complex on immobilized biological samples.

Such an approach could be applied to confocal immunofluorescencemicroscopy, which, using anti specific human MHC/pathogen-derivedpeptide complex antibodies, would permit analysis of the intracellularsite(s) of assembly and trafficking of such complexes. In situlocalization of APCs displaying or expressing specific humanMHC/pathogen-derived peptide complexes would be especially valuable incharacterizing the intercellular interactions between APCs and Tlymphocytes involved in initiation, propagation, and maintenance of antiviral T lymphocyte immune responses. Multicolor histochemistry could beused to reveal not only the type and location of viral APCs but also thephenotype of interacting anti viral T lymphocytes, including the set ofcytokines elicited.

The fact that 62 percent of the HLA-A2/Tax₁₁₋₁₉ complex binding Fab'swere peptide specific and MHC restricted was unexpected since theseantibodies were selected from a non immune repertoire considered not tobe biased toward such specificity. The fact that it was possible toisolate from the same phage library recombinant Fab's capable ofspecifically binding a large variety of specific MHC/peptide complexescomprising various cancer associated or viral HLA-A2 restricted peptides(Denkberg, G. et al., 2002. Proc. Natl. Acad. Sci. U.S.A. 99:9421-9426;Lev, A. et al., 2002. Cancer Res. 62:3184-3194) indicates that thecapacity to isolate anti MHC/Tax₁₁₋₁₉ complex antibodies from such alibrary was not Tax or peptide related. It is possible that oneparticular antibody family or antibody V gene segment could have anintrinsic propensity to bind HLA-A2 molecules, and that the highfrequency could be explained by a high abundance of such antibodies inthe non immune library. However, the sequences of the selected clonesare derived from many different antibody families and germline segments,without any biases visible in the complementarity determining regions(CDRs) either (Table 3). The high frequency and high affinities for someof the antibodies isolated herein suggest that these molecules may bepresent at a high frequency in the antibody repertoires from the B celldonors of the phage library, however an in-vivo role for such antibodiesremains unclear.

Whatever the reason for this high frequency of Fab's to bind specificMHC/peptide complexes may be, it appears that this phage based approachcan be successfully applied to identify recombinant antibodies capableof specifically binding to a large variety of specific humanMHC/pathogen-derived peptide complexes. Thus, it may now be possible toelucidate the role of pathogen derived antigens not only from theperspective of the T lymphocyte, using MHC/pathogen-derived peptidecomplex based TCR detection reagents such as tetrameric single chainMHC/pathogen-derived peptide complexes, but also from the perspective ofpathogen-derived APCs and diseased cells, using the novel antibody typedescribed herein.

A further application for anti specific human MHC/pathogen-derivedpeptide complex antibodies is in structure function studies of specificinteractions between such complexes and cognate TCRs. By mutatingparticular residues in the MHC restricted pathogen-derived peptide andtesting the influence of these mutations on the binding of the Fab's andpeptide specific T lymphocyte clones, it may be possible to obtainimportant data regarding the structure function relationship and thedifferent nature of the recognition process between such Fab's and thenative TCR (Stryhn A. et al., 1996. Proc. Natl. Acad. Sci. U.S.A.93:10338-10342).

CONCLUSIONS

By virtue of the capacity of the presently described Fab's tospecifically bind with optimal affinity and specificity particular humanAPM/pathogen-derived antigen complexes, such reagents are uniquelysuitable and optimal relative to all prior art compounds for: (a)identification, and characterization of cells/tissues expressing ordisplaying such complexes; (b) specific killing of cells displaying suchcomplexes by targeting cytotoxic drugs or radionuclides to pathogeninfected cells analogously to previously described methodologies (Boon,T. and van der Bruggen, P., 1996. J. Exp. Med. 183:725-729; Renkvist, N.et al., 2001. Cancer Immunol. Immunother. 50:3-15; Rosenberg, S. A.,2001. Nature 411:380-384); (c) confocal microscopic visualization andcharacterization of the intracellular localization and trafficking ofsuch complexes; (d) tracking of cells displaying such complexes inreal-time via confocal microscopy and in-vivo; and (e) modulation ofimmune responses by blocking interactions between specific humanAPM/pathogen-derived antigen complexes and cognate TCRs, analogously topreviously described methodologies practiced by the present inventors(Denkberg, G. et al., 2002. Proc. Natl. Acad. Sci. U.S.A. 99:9421-9426;Lev, A. et al., 2002. Cancer Res. 62:3184-3194). For example, thepresently described reagents could be used control pathogenic Tlymphocyte mediated anti pathogen immune responses without the risk ofantigen administration to an infected individual, and without the lossof function of an entire MHC allele, as would be the case with prior artanti MHC antibodies.

Thus, the presently described compounds are uniquely and optimallysuitable for diagnosing, characterizing and treating diseases in humanscaused by pathogens such as viruses, and for studying aspects of suchdiseases involving antigen presentation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent, patent application or sequence identified by itsaccession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

What is claimed is:
 1. A composition-of-matter comprising a solubleantibody including an antigen-binding region capable of specificallybinding a complex composed of a human antigen-presenting molecule and anantigen derived from a human T lymphotropic virus-1 (HTLV-1) TAXpolypeptide, wherein said human antigen-presenting molecule is a singlechain antigen-presenting molecule, wherein said human antigen-presentingmolecule comprises HLA-A2 molecule, wherein said binding of saidantibody to said complex is characterized by an affinity having adissociation constant from the range of 5×10⁻⁸ molar to 5×10⁻⁹ molar,wherein the antibody does not bind the human antigen-presenting moleculein an absence of the HTLV-1 TAX polypeptide, and wherein the antibodydoes not bind the HTLV-1 TAX polypeptide in an absence of the humanantigen-presenting molecule, and whereas a monovalent or divalentconfiguration of said soluble antibody binds a complex comprising saidantigen derived from HTLV-1 TX polypeptide when naturally presented on acell.
 2. The composition-of-matter of claim 1, wherein said antibody isa monoclonal antibody.
 3. The composition-of-matter of claim 1, whereinsaid antibody is of human origin.
 4. The composition-of-matter of claim1, further comprising a toxin or detectable moiety attached to saidantibody.
 5. The composition-of-matter of claim 1, wherein said antigenderived from said HTLV-1 TAX polypeptide is restricted by saidantigen-presenting molecule.